Methods of Diagnosing and Treating Cancer in Patients Having or Developing Resistance to a First Cancer Therapy

ABSTRACT

A method of identifying a subject having cancer who is likely to benefit from treatment with a combination therapy with a RAF inhibitor and a second inhibitor is provided. A method of treating cancer in a subject in need thereof is also provided and includes administering to the subject an effective amount of a RAF inhibitor and an effective amount of a second inhibitor, wherein the second inhibitor is a MEK inhibitor, a CRAF inhibitor, a CrkL inhibitor or a TPL2/COT inhibitor. A method of identifying a kinase target that confers resistance to a first inhibitor is also provided.

RELATED APPLICATIONS

This application claims the benefit of the filing date under 35 U.S.C.§119(e) of the following Provisional U.S. Patent Application Serial Nos.61/312,193, filed Mar. 9, 2010; 61/312,519, filed Mar. 10, 2010;61/326,021, filed Apr. 20, 2010; and 61/415,569, filed Nov. 19, 2010,all of which are hereby incorporated by reference in their entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under federal grantnumbers K08 CA115927 and 1DP20D002750 awarded by National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND

Oncogenic mutations in the serine/threonine kinase B-RAF (also known asBRAF) are found in 50-70% of malignant melanomas. (Davies, H. et al.,Nature 417, 949-954 (2002).) Pre-clinical studies have demonstrated thatthe B-RAF(V600E) mutation predicts a dependency on the mitogen-activatedprotein kinase (MAPK) signalling cascade in melanoma (Hoeflich, K. P. etal., Cancer Res. 69, 3042-3051 (2009); McDermott, U. et al., Proc. Natl.Acad. Sci. USA 104, 19936-19941 (2007); Solit, D. B. et al. BRAFmutation predicts sensitivity to MEK inhibition. Nature 439, 358-362(2006); Wan, P. T. et al., Cell 116, 855-867 (2004); Wellbrock, C. etal., Cancer Res. 64, 2338-2342 (2004))—an observation that has beenvalidated by the success of RAF or MEK inhibitors in clinical trials(Flaherty, K. T. et al., N. Engl. J. Med. 363, 809-819 (2010); Infante,J. R. et al., J. Clin. Oncol. 28 (suppl.), 2503 (2010); Schwartz, G. K.et al., J. Clin. Oncol. 27 (suppl.), 3513 (2009).) However, clinicalresponses to targeted anticancer therapeutics are frequently confoundedby de novo or acquired resistance. (Engelman, J. A. et al., Science 316,1039-1043 (2007); Gorre, M. E. et al., Science 293, 876-880 (2001);Heinrich, M. C. et al., J. Clin. Oncol. 24, 4764-4774 (2006); Daub, H.,Specht, K. & Ullrich, A. Nature Rev. Drug Discov. 3, 1001-1010 (2004).)Accordingly, there remains a need for new methods for identification ofresistance mechanisms in a manner that elucidates “druggable” targetsfor effective long-term treatment strategies, for new methods ofidentifying patients that are likely to benefit from the treatmentstrategies, and for methods of treating patients with the effectivelong-term treatment strategies.

BRIEF SUMMARY

The present invention relates to the development of resistance totherapeutic agents in the treatment of cancer and identification oftargets that confer resistance to treatment of cancer. The presentinvention also relates to identification of parallel drug targets forfacilitating an effective long-term treatment strategy and toidentifying patients that would benefit from such treatment.

Accordingly, in one aspect, a method of identifying a subject havingcancer who is likely to benefit from treatment with a combinationtherapy with a RAF inhibitor and a second inhibitor is provided. Themethod includes assaying a gene copy number, a mRNA or a protein levelor phosphorylation of one or more kinase targets selected from the groupconsisting of MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL (CrkL), FGR (Fgr),PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3)in cancer cells obtained from the subject. The method further includescomparing the gene copy number, the mRNA or the protein level or thephoshorylation with a gene copy number, a mRNA or a protein level orphosphorylation of the target kinase in cells obtained from a subjectwithout the cancer and correlating increased gene copy number or analteration in mRNA expression or protein overexpression orphosphorylation of the target kinase in the cancer cells relative to thecells from the subject without the cancer with the subject having thecancer who is likely to benefit from treatment with the combinationtherapy.

In another aspect, a method of treating cancer in a subject in needthereof is provided. The method includes administering to the subject aneffective amount of a RAF inhibitor and an effective amount of a secondinhibitor, wherein the second inhibitor is a MEK inhibitor or a TPL2/COTinhibitor.

In another aspect, a method of identifying a kinase target that confersresistance to a first inhibitor is provided. The method includesculturing cells having sensitivity to the first inhibitor and expressinga plurality of kinase ORF clones in the cell cultures, each cell cultureexpressing a different kinase ORF clone. The method further includesexposing each cell culture to the inhibitor and identifying cellcultures having greater viability than a control cell culture afterexposure to the inhibitor to identify the kinase ORF clone that confersresistance to the first inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an ORF-based functional screen which identified COTand C-RAF kinases as drivers of resistance to B-RAF inhibition. (a)Schematic overview of the CCSB/Broad Institute Kinase ORF collection.Kinase classification and number of kinases per classification arenoted; (b) A375 cells expressing the CCSB/Broad Institute Kinase ORFcollection were assayed for relative viability in 1 μM PLX4720 andnormalized to constitutively active MEK1 (MEK1 DD). Nine ORFs (circles)scored 2 standard deviations (dashed line, 58.64%) from the mean of allORFs (dashed line, 44.26%); (c) Indicated ORFs were expressed in 5B-RAF^(V600E) cell lines and treated with DMSO or 1 μM PLX4720.Viability (relative to DMSO) was quantified after 4 days. Error barsrepresent standard deviation between replicates (n=6); (d) Secondaryscreen in A375 and SKMEL28 prioritizes the top 9 candidate ORFs across amultipoint PLX4720 concentration scale.

FIG. 2 illustrates resistance to B-RAF inhibition via MAPK pathwayactivation. (a) Indicated ORFs were expressed in A375. Levels ofphosphorylated MEK and ERK were assayed following 18 h. treatment withDMSO (−) or PLX4720 (concentration noted); (b) Proliferation of A375expressing indicated ORFs. Error bars represent standard deviationbetween replicates (n=6). (c) C-RAF (S338) and ERK phosphorylation inlysates from A375 expressing indicated ORFs. (d) COT expression inlysates from immortalized primary melanocytes expressing BRAFV600E orempty vector. COT mRNA has an internal start codon (30M) resulting intwo protein products of different lengths; amino acids 1-467 or 30-467,noted with arrows. (e) COT and ERK phosphorylation in lysates from A375expressing indicated ORFs following shRNA-mediated B-RAF depletion(shBRAF) relative to control shRNA (shLuc). (f) ERK phosphorylation inlysates from A375 expressing indicated ORFs following shRNA-mediatedC-RAF depletion (shCRAF) or control shRNA (shLuc), following 18 h.treatment with DMSO (−) or 1 μM PLX4720 (+).

FIG. 3 illustrates COT expression predicts resistance to B-RAFinhibition in cancer cell lines. (a) MAP3K8/COT copy number; red bars:COT amplification, blue bars: non-amplified COT; (b) COT expression inB-RAF^(V600E) cell lines and (c) short-term cultures; (d) PLX4720 GI50in B-RAF^(V600E) cell lines. Colors as in (a); (e) MEK and ERKphosphorylation following treatment with DMSO or PLX4720 (concentrationindicated); (f) ERK phosphorylation in M307 lysates (AZD-R;AZD6244-resistant) treated with DMSO or 1 μM PLX4720 (PLX) or CI-1040(CI); (g) COT mRNA expression (QRT/PCR) in patient/lesion-matchedPLX4032-treated metastatic melanoma tissue samples. Pts. 1 and 3 hadmultiple biopsies from the same lesion. Error bars represent SEM (n=3)U; undetermined/undetectable; (h) ERK and MEK phosphorylation inRPMI-7951 following shRNA-mediated COT depletion (shCOT) versus control(shLuc) and treatment with DMSO (−) or 1 μM PLX4720 (+). ERK and MEKphosphorylation are quantified; (i) ERK and MEK phosphorylation inRPMI-7951 following 1 h. treatment with a small molecule COT kinaseinhibitor. ERK and MEK phosphorylation are quantified. (j) PLX4720sensitivity curves in a panel of BRAF^(V600E) cell lines. OUMS-23 andM307 represent cell lines with COT expression/amplification and allothers represent cell lines with undetectable/unaltered COT; (k)Selective expression of COT and corresponding MAPK pathway activation ina metastatic subcutaneous malignant melanoma with acquired resistance toPLX4032 (* denotes a background band, MET-MM (PLX-R); metastaticmalignant melanoma, PLX4032-resistant).

FIG. 4 illustrates COT-expressing B-RAFV600E cell lines exhibitresistance to allosteric MEK inhibitors. (a) CI-1040 GI50 in a panel ofB-RAF^(V600E) cell lines; (b) MEK and ERK phosphorylation in lysatesfrom indicated cell lines treated with DMSO or CI-1040 (concentrationnoted); (c) Fold change (relative to MEK1) GI50 of A375 ectopicallyexpressing the indicated ORFs for PLX4720, RAF265, CI-1040 and AZD6244;(d) ERK phosphorylation in A375 expressing indicated ORFs followingtreatment with DMSO or 1 μM of PLX4720, RAF265, CI-1040 or AZD6244; (e)Viability of A375 expressing the indicated ORFs and treated with DMSO,PLX4720 (concentration indicated) and PLX4720 in combination withCI-1040 or AZD6244 (all 1 μM). Error bars represent the standarddeviation (n=6); (f) ERK phosphorylation in A375 expressing indicatedORFs following treatment with DMSO, PLX4720 (1 μM) or PLX4720 incombination with CI-1040 or AZD6244 (all 1 μM); (g) Cell lines withaberrant MAP3K8/COT copy number/expression are insensitive to theallosteric MEK inhibitor CI-1040 or (h) AZD6244; (i) A schematicoutlining the formation of MAP3K complexes in response to B-RAFinhibition in B-RAF^(V600E)-mutant cell lines. PLX4720 positions C-RAFin a signaling-competent complex (upper right panel) that is activatedby oncogenic events upstream of C-RAF (lower right panel), subsequentlydriving resistance. In the context of COT expression, COT/RAF-containingcomplexes are sufficient to activate the MAPK pathway and mediateresistance (lower left panel).

FIG. 5 illustrates a schematic outline of an ORF-based functional screenfor kinases that drive resistance to B-RAF inhibition. The B-RAF^(V600E)cell line A375 was lentivirally transduced with the 597 kinases in theCCSB/Broad Institute Kinase ORF Collection. ORFs having a positive ornegative effect on proliferation in control-treated A375 were identifiedand removed from final analysis. Resistance-promoting ORFs wereidentified by generating a differential viability ratio between B-RAFinhibited (PLX4720-treated) and control-treated cells. Differentialviability was normalized to a constitutively active MEK1 allele,MEK1^(DD); an assay specific positive control.

FIG. 6 illustrates that the CCSB/Broad Institute Kinase ORF Collectionis well expressed via high titer lentivirus. (a), schematic of thepLX-BLAST-V5 lentiviral expression vector used for all ORF-screens andsubsequent validation. (b) GFP-tagged ORFs representing a broad sizerange were lentivirally expressed in Jurkat cells and the percentage ofGFP-expressing cells/ORF (e.g., infected cells) quantified,demonstrating high viral titer across a range of ORF sizes. (c),expression of 96 random ORFs detected via LiCor with antibodies againstthe V5-epitope tag, relative to cellular DNA. Expression was detectablein 83% of the wells.

FIG. 7 illustrates the expression of candidate resistance ORFs. 293Twere transiently transfected with pLX-BLAST-V5-ORF (indicated) andexpression detected using an anti-V5-HRP antibody. The AXL clone is‘closed’ and has a stop codon preceding the V5 tag. See FIG. 12 forverification of expression; (*) on dark-exposure indicate the expressionof three ORFs not visible in the lighter exposure.

FIG. 8 illustrates that a secondary screen prioritizes the top 9candidate B-RAF inhibitor resistance ORFs. The top nine ORFs scoring inthe primary screen were expressed in A375 or SKMEL28 and a GI₅₀ from an8-point PLX4720 concentration range.

FIG. 9 illustrates the effects of ORF expression on proliferation inB-RAF^(V600E) cell lines. Proliferation, relative to MEK1, in (a) A375or (b) SKMEL28 expressing indicated ORFs after 7 days of growth.

FIG. 10 illustrates that ectopic expression of constitutively activeMEK1 (MEK1^(DD)) and COT lead to increased pMEK/pERK in A375, whereasC-RAF reduces pMEK/pERK levels. Lysates from A375 ectopically expressingGFP, MEK1, MEK^(DD), COT or C-RAF were analyzed via immunoblot forlevels of pERK and pMEK. GFP and MEK1 (lanes 1-3) were separated fromCOT/C-RAF (lanes 4-5) to prevent residual V5-MEK1 signal fromoverwhelming that of COT and C-RAF, which are expressed at much lowerlevels.

FIG. 11 illustrates that the kinase activity of COT and C-RAF isrequired for sustained ERK phosphorylation in the context of PLX4720treatment. Immunoblot analysis of A375 expressing ectopic (a) MEK1, wildtype COT or kinase inactive COT (COT^(K167R)) or (b) MEK1, wild typeC-RAF or kinase inactive C-RAF (C-RAF^(K375M)) treated with 1 μM PLX4720for 18 h.

FIG. 12 illustrates the effects of ORF expression on MAPK signaling inthe context of the B-RAF inhibitor PLX4720. MAPK pathway activation wasassessed by immunoblot analysis of pERK and pMEK in A375 expressing theindicated ORFs in the presence of PLX4720 (18 h., concentrationindicated). (*) indicates the use of an antibody directed against theexpressed ORF, not the V5 epitope. AXL was cloned without the V5 tag.

FIG. 13 illustrates that B-RAF associates with immunoprecipitated C-RAFin A375 following 18 hr. treatment with 1 μM PLX4720 (+) or DMSO (−),(a). WCE; whole cell extract controls. Ectopically expressed C-RAFconstitutively associates with B-RAF and is phosphorylated at S338,consistent with membrane localization and activation. MEK1, MEK^(DD) andCOT-expressing A375 show no evidence of C-RAF activation, (b).

FIG. 14 illustrates that Retroviral expression of a wild-type C-RAF or ahigh-activity truncation mutant of C-RAF (C-RAF(22W)) renders A375resistant to the B-inhibitor PLX4720 (a) and leads to sustained pERKlevels in the context of PLX4720 treatment (1 μM, 18 h.), (b). C-RAFexpression levels achieved with retroviruses are significantly lowerthan with lentiviral-based systems, resulting in a lower GI₅₀ than thatachieved with lentiviral C-RAF.

FIG. 15 illustrates the effects of B-RAF^(V600E) on COT mRNA (a)Quantitative RT/PCR of COT mRNA expression relative to GAPDH mRNAexpression in transformed primary melanocytes expressing wild-type B-RAF(vector) or B-RAF^(V600E) COT expression was normalized to that ofvector-expressing primary melanocytes. (**) Significant, p 0.05(Student's two-tailed, paired T-Test). Endogenous COT mRNA isundetectable in PLX4720-sensitive A375 and ectopically expressed COTmRNA levels are unaffected by 1 μM PLX4720 treatment. A375 expressingGFP or COT were treated for 18 h. with 1 μM PLX4720. Reverse-transcribedmRNA was analyzed for GAPDH-normalized COT expression, relative toGFP-expressing, DMSO treated, A375. (*) Not significant, p>0.05(Student's two-tailed, paired T-Test). Error bars represent SEM.

FIG. 16 illustrates that B- and C-RAF protein levels are not requiredfor COT-mediated ERK phosphorylation. A375 expressing ectopic MEK1(control) or COT were sequentially infected with lentivirus expressingshRNAs targeting B-RAF, C-RAF or control shRNA (shLuc) and assayed forexpression of the indicated proteins in the presence (+) or absence (−)of 1 μM PLX4720, 18 h.

FIG. 17 illustrates SNP analysis of 752 cell lines reveals copy numberalterations in MAP3K8/COT. Of the 752 cell lines hat had undergone copynumber analysis, 534 had also undergone mutation profiling. Thirty-eight(7.1%) of mutation-profiled cells harbor the B-RAF^(V600E) mutation. Twocell lines (OUMS-23, RPMI-7951, indicated) harbor the B-RAF^(V600E)mutation along with copy number gain in MAP3K8/COT.

FIG. 18 illustrates MAP3K8/COT alterations in the cancer cell lineOUMS-23. (a) RMA signal of a MAP3K8/COT probe (noted) from mRNAmicroarray analysis. OUMS-23 is one of the top 2% (of 765 cell lines)expressing COT mRNA. (b) COT mRNA expression in a panel ofB-RAF^(V600E)-mutant cell lines. (c) Endogenous COT protein expressionin OUMS-23 relative to ectopically expressed COT in A375 and SKMEL28cell lines, as determined via immunoblot analysis of the indicatedcells.

FIG. 19 illustrates that COT mRNA and protein are expressed in BRAF-inhibitor resistant cell lines and tissue. (a) RT/PCR analysis ofGAPDH normalized COT mRNA expression in a panel of cell lines, shortterm cultures and tissue from relapsed, PLX4032-treated, malignantmelanoma (MM-R). Corresponding protein expression for cell lines andshort term cultures are shown in FIGS. 3 b and 3 c, respectively. (b)Western blot analysis of lysates from primary melanocytes (1° Mel(B-RAFWT)), patient matched normal skin (Skin) and metastatic malignantmelanoma (MM-R; COT mRNA shown in panel a), A375 cells and primarymelanocytes expressing B-RAF^(V600E)(1° MeI (B-RAFV600E)).

FIG. 20 illustrates that depletion of COT affects viability in the COTamplified cell line RPMI-7951. (a) Quantification of RPMI-7951 viabilityfollowing lentiviral shRNA-mediated COT depletion (shCOT), relative tocontrol shRNA (shLuc). Error bars represent standard deviation betweenreplicates. (b) Immunoblot analysis showing relative COT proteinexpression in shLuc and shCOT-expressing RPMI-7951.

FIG. 21 illustrates effects of ORF expression on the GI₅₀ of a panel ofMAPK pathway inhibitors in SKMEL28. The half-maximal growth-inhibitoryconcentration (GI₅₀) of SKMEL28 ectopically expressing MEK1, MEK1^(DD)or COT was determined for the RAF inhibitors PLX4720 and RAF265 and theMEK1/2 inhibitors CI-1040 and AZD6244. The change in GI₅₀ for MEK1^(DD)and COT (relative to MEK1) was determined for each compound.

FIG. 22 illustrates that COT can activate ERK via MEK-independent andMEK-dependent mechanisms. (a) Immunoblot analysis of ERK phosphorylationin lysates from A375 following expression of GFP or COT and subsequentlentiviral shRNA-mediated MEK1, MEK2 or MEK1 and MEK2 (MEK1+2)depletion, relative to control shRNA (shLuc). Left and right panelsrepresent two different pairs of MEK1 and MEK2 shRNA constructs. (b)Immunoblot blot analysis of recombinant, inactive ERK phosphorylation(Thr202/Tyr204) by recombinant COT in an in vitro kinase assay.

FIG. 23 illustrates that combinatorial MAPK pathway inhibitioneffectively suppresses proliferation in SKMEL28. Viability (relative toDMSO) of SKMEL28 ectopically expressing MEK1, MEK1^(DD) or COT andtreated with DMSO, PLX4720 (concentration indicated), PLX4720 (1 μM) andCI-1040 (1 μM) or PLX4720 (1 μM) and AZD6244 (1 μM). Error barsrepresent the standard deviation between replicates.

FIG. 24 illustrates that COT over expression is sufficient to rendermelanoma cancer cells with the B-RAF^(V600E) mutation resistant to B-RAFinhibition.

FIG. 25 illustrates the top nine ORFs scoring in the primary screen wereexpressed in (a) SKEL28 of (b) A375 and a GI50 is shown for 4 MAPKpathway inhibitors (PLX4720, RAF265, CI-1040, AZD-6244).

FIG. 26 illustrates that CRKL expression modifies sensitivity to theselective B-RAF inhibitor PLX4720 in a panel of B-RAFv600E cell lines.

FIG. 27 illustrates the MAP3K8/COT-amplified/B-RAF^(V600E)-mutant cancercell line OUMS-23 shows constitutive phosphorylation of ERK/MEK across adose range of PLX4720.

FIG. 28 illustrates the insensitivity to MAPK pathway inhibitioncorresponds with MAP3K8/COT copy number gains in a subset of skin cancercell lines. A panel of 20 B-RAF^(V600E)-mutant cell lines and theirsensitivity to (a) the B-RAF inhibitor PLX4720 and (b) the MEK inhibitorAZD6244 is shown.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the development of resistance totherapeutic agents in the treatment of cancer and identification oftargets that confer resistance to treatment of cancer. The presentinvention also relates to identification of parallel drug targets forfacilitating an effective long-term treatment strategy and toidentifying patients that would benefit from such treatment. In someembodiments, the present invention relates to kinases and in particularto MAP kinase pathway components.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, immunology,microbiology, cell biology and recombinant DNA, which are within theskill of the art. See e.g., Sambrook, Fritsch and Maniatis, MOLECULARCLONING: A LABORATORY MANUAL, (Current Edition); CURRENT PROTOCOLS INMOLECULAR BIOLOGY (F. M. Ausubel et al. eds., (Current Edition)); theseries METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICALAPPROACH (Current Edition) ANTIBODIES, A LABORATORY MANUAL and ANIMALCELL CULTURE (R. I. Freshney, ed. (1987)). DNA Cloning: A PracticalApproach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N.Gait, ed., Current Edition); Nucleic Acid Hybridization (B. Hames & S.Higgins, eds., Current Edition); Transcription and Translation (B. Hames& S. Higgins, eds., Current Edition); Fundamental Virology, 2nd Edition,vol. I & II (B. N. Fields and D. M. Knipe, eds.)

The mitogen-activated protein kinase (MAPK) cascade is a criticalintracellular signaling pathway that regulates signal transduction inresponse to diverse extracellular stimuli, including growth factors,cytokines, and proto-oncogenes. Activation of this pathway results intranscription factor activation and alterations in gene expression,which ultimately lead to changes in cellular functions including cellproliferation, cell cycle regulation, cell survival, angiogenesis andcell migration. Classical MAPK signaling is initiated by receptortyrosine kinases at the cell surface, however many other cell surfacemolecules are capable of activating the MAPK cascade, includingintegrins, heterotrimeric G-proteins, and cytokine receptors.

Ligand binding to a cell surface receptor, e.g., a receptor tyrosinekinase, typically results in phosphorylation of the receptor. Theadaptor protein Grb2 associates with the phosphorylated intracellulardomain of the activated receptor, and this association recruits guaninenucleotide exchange factors including SOS-I and CDC25 to the cellmembrane. These guanine nucleotide exchange factors interact with andactivate the GTPase Ras. Common Ras isoforms include K-Ras, N-Ras, H-Rasand others. Following Ras activation, the serine/threonine kinase Raf(e.g., A-Raf, B-Raf or Raf-1) is recruited to the cell membrane throughinteraction with Ras. Raf is then phosphorylated. Raf directly activatesMEKI and MEK2 by phosphorylation of two serine residues at positions 217and 221. Following activation, MEKI and MEK2 phosphorylate tyrosine(Tyr-185) and threonine (Thr-183) residues in serine/threonine kinasesErkl and Erk2, resulting in Erk activation. Activated Erk regulates manytargets in the cytosol and also translocates to the nucleus, where itphosphorylates a number of transcription factors regulating geneexpression. Erk kinase has numerous targets, including Elk-I, c-Etsl,c-Ets2, p90RSKI, MNKI, MNK2, MSKI, MSK2 and TOB. While the foregoingpathway is a classical representation of MAPK signaling, there isconsiderable cross talk between the MAPK pathway and other signalingcascades.

Aberrations in MAPK signaling have a significant role in cancer biology.Altered expression of Ras is common in many cancers, and activatingmutations in Ras have also been identified. Such mutations are found inup to 30% of all cancers, and are especially common in pancreatic (90%)and colon (50%) carcinomas. In addition, activating Raf mutations havebeen identified in melanoma and ovarian cancer. The most commonmutation, BRAF^(V600E), results in constitutive activation of thedownstream MAP kinase pathway and is required for melanoma cellproliferation, soft agar growth, and tumor xenograft formation. Based onthe defined role of MAPK over-activation in human cancers, targetingcomponents of the MAPK pathway with specific inhibitors is a promisingapproach to cancer therapy. However, patients may have innate resistanceor acquire resistance to these promising therapies. Identification oftarget kinases, diagnostic and/or prognostic markers and treatmenttherapies for these patients with innate or acquired resistance aredescribed below.

High Throughput Functional Screening Assay

In some aspects, the present invention relates to methods of identifyingtargets capable of driving resistance to clinically efficacioustherapies using a high throughput screening assay. In some embodiments,the method may include an open reading frame (ORF)-based functionalscreen for kinases that drive resistance to therapeutic agents. Themethod may include providing a cell line with a kinase known to have anoncogenic mutation. A library of kinase ORFs may be individuallyexpressed in the cell line so that a plurality of clones each expressinga different ORF from the library may be further evaluated. Each clonemay be (1) exposed to an inhibitor of the known kinase in the cell lineand (2) monitored for growth changes based on the expression of the ORFin the cell line without the inhibitor. Any clones having a growtheffect from the ORF expression alone, either positive or negativegrowth, are eliminated. The remaining clones each expressing a differentkinase are then compared for viability between a control and a treatedclone and normalized to a positive control. Increased cell viabilityafter treatment with the inhibitor identifies ORFs that conferresistance and therefore identifies kinase targets for treatment with anadditional inhibitor. In some embodiments, clones scoring above twostandard deviations from the normalized mean may be target kinasesindicating treatment with an additional inhibitor is beneficial to thesubject.

By way of non-limiting example, a schematic of a high throughputfunctional screening assay for kinases that drive resistance to B-RAFinhibition is shown in FIG. 5. A collection of ˜600 cloned and sequencevalidated ORFs were assembled, accounting for ˜75% of all annotatedkinases (Center for Cancer Systems Biology (CCSB)/Broad Institute KinaseORF Collection, FIGS. 1 a, 1 b, Table 3). This publically availablecollection can be rapidly transferred into a variety of expressionvectors for various end-applications. Any type of expression vectorknown to one skilled in the art may be used to express the Kinase ORFcollection. By way of non-limiting example, a selectable,epitope-tagged, lentiviral expression vector capable of producing hightiter virus and robust ORF expression in mammalian cells may be createdto express the kinase collection, (pLX-BLAST-V5, FIG. 6 a).

To identify kinases capable of circumventing RAF inhibition, the arrayedkinase ORF collection may be stably expressed in A375, a B-RAF^(V600E)malignant melanoma cell line that is sensitive to the RAF kinaseinhibitor PLX4720 (FIGS. 1 a, 1 b and 6 c, Table 3). Clones of ORFexpressing cells treated with 1 μM PLX4720 are screened for viabilityrelative to untreated cells and normalized to an assay-specific positivecontrol, MEK1^(S218/222D) (MEK1^(DD)) (Table 4). ORFs that affectedbaseline viability or proliferation are removed from the analysis.Clones scoring above two standard deviations from the normalized meanmay be further evaluated to identify a resistance conferring targetkinase for a second inhibitor. In some embodiments, the gene encodingthe target kinase may be MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL (CrkL),FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), orPAK3 (Pak3). In some embodiments, the gene encoding the target kinasemay be a MAPK pathway activator. In some embodiments, the gene encodingthe target kinase may be a MAP3 kinase that directly phosphorylates andactivates MEK. In some embodiments, the gene encoding the target kinasemay encode an adapter protein that is amplified and phosphorylated inmelanoma.

In other embodiments, the ORF collection may be stably expressed in acell line having a different mutation in B-RAF, for example, anothermutation at about amino acid position 600 such as V600K, V600D, andV600R. Additional B-RAF mutations include the mutations described inDavies et al. Nature, 417, 949-954, 2002, Table 1. Cell lines may beused that are sensitive to other RAF kinase inhibitors including, butnot limited to, PLX4032; GDC-0879; RAF265; sorafenib; SB590855 and/or ZM336372. In some embodiments, the ORF collection may be stably expressedin a cell line having a sensitivity to a MEK inhibitor. Non-limitingexamples of MEK inhibitors include, AZD6244; CI-1040; PD184352;PD318088, PD98059, PD334581, RDEA119,6-Methoxy-7-(3-morpholin-4-yl-propoxy)-4-(4-phenoxy-phenylamino)-quinoline-3-carbonitrileand4-[3-Chloro-4-(1-methyl-1H-imidazol-2-ylsulfanyl)-phenylamino]-6-methoxy-7-(3-morpholin-4-yl-propoxy)-quinoline-3-carbonitrile.Additional RAF and MEK inhibitors are described below. By way ofnon-limiting example, exemplary RAF inhibitors are shown in Table 1 andexemplary MEK inhibitors are shown in Table 2.

TABLE 1 Exemplary RAF Inhibitors Name CAS No. Structure 1 RAF265 927880-90-

2 Sorafenib Tosylate Nexavar Bay 43-9006 475207- 59-1

Sorafenib 4-[4-[[4-chloro-3- (trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methyl-pyridine-2- carboxamide 284461- 73-0

3 SB590885

4 PLX4720 918505- 84-7

5 PLX4032 1029872- 54-5

6 GDC-0879 905281- 76-7

7 ZM 336372 208260- 29-1

TABLE 2 Exemplary MEK Inhibitors Name CAS No. Structure 1CI-1040/PD184352 212631-79-3

2 AZD6244 606143-52-6

3 PD318088 391210-00-7

4 PD98059 167869-21-8

5 PD334581

6 RDEA119 N-[3,4-difluoro-2-[(2-fluoro- 4-iodophenyl)amino]-6-methoxyphenyl]-1-[(2R)- 2,3-dihydroxypropyl]- Cyclopropanesulfonamide923032-38-6

7 6-Methoxy-7-(3-morpholin- 4-yl-propoxy)-4-(4-phenoxy-phenylamino)-quinoline-3- carbonitrile

8 4-[3-Chloro-4-(1-methyl-1H- imidazol-2-ylsulfanyl)-phenylamino]-6-methoxy-7- (3-morpholin-4-yl-propoxy)-quinoline-3-carbonitrile

Diagnostic/Prognostic Markers for Innate and Acquired Resistance toTargeted Therapies

In some aspects, the present invention relates to methods of detectingthe presence of one or more diagnostic or prognostic markers in a sample(e.g. a biological sample from a cancer patient). A variety of screeningmethods known to one of skill in the art may be used to detect thepresence of the marker in the sample including DNA, RNA and proteindetection. The techniques described below can be used to determine thepresence or absence of a kinase target in a sample obtained from apatient. In some embodiments, the patient may have innate or acquiredresistance to kinase targeted therapies, including B-RAF inhibitors orMEK inhibitors. For example, the patient may have an innate or acquiredresistance to B-RAF inhibitors PLX4720 and/or PLX4032. In someembodiments, the patient may have innate or acquired resistance to MEKinhibitor AZD6244. Identification of one or more kinase targets markersin a patient assists the physician in determining a treatment protocolfor the patient. For example, in a patient having one or more kinasetarget markers, the physician may treat the patient with a combinationtherapy as described in more detail below.

In some embodiments, the kinase target may include, but is not limitedto, MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL (CrkL), FGR (Fgr), PRKCE(Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3). Themarker may be an increase in the gene copy number, an increase inprotein expression, phosphorylation of one or more MAP kinase pathwaymembers, a change in mRNA expression and the like, for the kinasetarget.

By way of non-limiting example, in a patient having an oncogenicmutation in B-RAF, identification of an activated target kinase can beuseful for characterizing a treatment protocol for the patient. Forexample, in a patient having a B-RAF^(V600E) mutation, treatment with aRAF inhibitor alone may indicate that the patient is at relatively highrisk of acquiring resistance to the treatment after a period of time. Ina patient having an oncogenic mutation, identification of an activatedkinase target in that patient may indicate inclusion of a secondinhibitor in the treatment protocol.

Identification of an activated kinase target may include an analysis ofa gene copy number and identification of an increase in copy number of atarget kinase. For example, a copy number gain in MAP3K8 is indicativeof a patient having innate resistance or developing acquired resistance,in particular if the patient also has a B-RAF^(V600E) mutation.

In some embodiments, identification of an activated kinase target mayinclude an analysis of phosphorylation of a kinase target and/or amember of the MAP kinase pathway. For example, phosphorylation of C-RAFat S338 is indicative of a patient having innate resistance ordeveloping acquired resistance, in particular if the patient also has aB-RAF^(V600E) mutation. In some embodiments, identification of anincrease in MEK/ERK phosphorylation may be indicative of a patienthaving innate resistance or developing acquired resistance. IncreasedCOT protein expression in patients having a B-RAF^(V600E) mutation maypredict resistance to RAF inhibition and MEK inhibition.

Identification of an activated kinase target may include an analysis ofmRNA expression of a kinase target. For example, an increase in COT mRNAexpression following initial treatment with a first kinase inhibitor isindicative of a patient having or developing resistance. In someembodiments, the first kinase inhibitor may be a RAF inhibitor or a MEKinhibitor.

Methods of Treatment

In various embodiments, the invention provides methods for treatment ofa patient having cancer. The methods generally comprise administrationof a first inhibitor and a second inhibitor. One inhibitor may be a RAFinhibitor. The RAF inhibitor may be a pan-RAF inhibitor or a selectiveRAF inhibitor. Pan-RAF inhibitors include but are not limited to RAF265,sorafenib, or SB590885. In some embodiments, the RAF inhibitor is aB-RAF inhibitor. In some embodiments, the selective RAF inhibitor isPLX4720, PLX4032, or GDC-0879-A. One inhibitor may be a MEK inhibitor(see Table 2 illustrating exemplary MEK inhibitors). One inhibitor maybe a COT inhibitor. By way of non-limiting example, the COT inhibitormay be a shRNA inhibitor as described below or a small molecule COTinhibitor,4-(3-chloro-4-fluorophenylamino)-6-(pyridin-3-yl-methylamino)-3-cyano-[1,7]-naphthyridine(EMD; TPL2 inhibitor I; catalogue number 616373, PubChem ID: 9549300)Inhibitors of the present invention inhibit one or more of the kinasetargets including MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL (CrkL), FGR(Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3(Pak3) or other MAP kinase pathway targets.

In some embodiments, a combination therapy for cancer is provided,comprising an effective amount of a RAF inhibitor and an effectiveamount of a MAP3K8 (TPL2/COT) inhibitor. Also provided herein is acombination therapy for cancer, comprising an effective amount of a RAFinhibitor and an effective amount of a MEK inhibitor. Other combinationtherapies include an effective amount of a RAF inhibitor and aneffective amount of a second inhibitor targeting the gene, mRNA orprotein encoded by one or more of the following: MAP3K8 (TPL2/COT), RAF1(CRAF), CRKL (CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2(ErbB2), AXL (Axl), or PAK3 (Pak3). The combination therapy is suitablefor treatment of a patient wherein the cancer contains B-RAF mutantcells and in particular, B-RAF^(V600E) mutant cells. The presentinvention further provides a combination therapy for cancer, comprisingan effective amount of a RAF inhibitor and an effective amount of a MEKinhibitor, wherein the subject with the cancer contains cells withaltered MAP3K8 (TPL2/COT) expression or gene copy number. In someembodiments, the MEK inhibitor is CI-1040/PD184352 or AZD6244.

As a non-limiting example, the MEK inhibitor provided herein can beCI-1040, AZD6244, PD318088, PD98059, PD334581, RDEA119,6-Methoxy-7-(3-morpholin-4-yl-propoxy)-4-(4-phenoxy-phenylamino)-quinoline-3-carbonitrileor4-[3-Chloro-4-(1-methyl-1H-imidazol-2-ylsulfanyl)-phenylamino]-6-methoxy-7-(3-morpholin-4-yl-propoxy)-quinoline-3-carbonitrile,Roche compound RG7420, or combinations thereof. Additional MEKinhibitors known in the art may also be used.

In exemplary embodiments of the foregoing aspects, the RAF inhibitorprovided herein is PLX4720, PLX4032, BAY 43-9006 (Sorafenib), ZM 336372,RAF 265, AAL-881, LBT-613, or CJS352 (NVP-AAL881-NX (hereafter referredto as AAL881) and NVP-LBT613-AG-8 (LBT613) are isoquinoline compounds(Novartis, Cambridge, Mass.). Additional exemplary RAF inhibitors usefulfor combination therapy include pan-RAF inhibitors, inhibitors of B-RAF,inhibitors of A-RAF, and inhibitors of RAF-1. In exemplary embodimentsRAF inhibitors useful for combination therapy include PLX4720, PLX4032,BAY 43-9006 (Sorafenib), ZM 336372, RAF 265, AAL-881, LBT-613, andCJS352. Exemplary RAF inhibitors further include the compounds set forthin PCT Publication No. WO/2008/028141, the entire contents of which areincorporated herein by reference. Exemplary RAF inhibitors additionallyinclude the quinazolinone derivatives described in PCT Publication No.WO/2006/024836, and the pyridinylquinazolinamine derivatives describedin PCT Publication No. WO/2008/020203, the entire contents of which areincorporated herein by reference.

Administration of the combination includes administration of thecombination in a single formulation or unit dosage form, administrationof the individual agents of the combination concurrently but separately,or administration of the individual agents of the combinationsequentially by any suitable route. The dosage of the individual agentsof the combination may require more frequent administration of one ofthe agents as compared to the other agent in the combination. Therefore,to permit appropriate dosing, packaged pharmaceutical products maycontain one or more dosage forms that contain the combination of agents,and one or more dosage forms that contain one of the combinations ofagents, but not the other agent(s) of the combination.

Agents may contain one or more asymmetric elements such as stereogeniccenters or stereogenic axes, e.g., asymmetric carbon atoms, so that thecompounds can exist in different stereoisomeric forms. These compoundscan be, for example, racemates or optically active forms. For compoundswith two or more asymmetric elements, these compounds can additionallybe mixtures of diastereomers. For compounds having asymmetric centers,it should be understood that all of the optical isomers and mixturesthereof are encompassed. In addition, compounds with carbon-carbondouble bonds may occur in Z- and E-forms; all isomeric forms of thecompounds are included in the present invention. In these situations,the single enantiomers (optically active forms) can be obtained byasymmetric synthesis, synthesis from optically pure precursors, or byresolution of the racemates. Resolution of the racemates can also beaccomplished, for example, by conventional methods such ascrystallization in the presence of a resolving agent, or chromatography,using, for example a chiral HPLC column.

Unless otherwise specified, or clearly indicated by the text, referenceto compounds useful in the combination therapy of the invention includesboth the free base of the compounds, and all pharmaceutically acceptablesalts of the compounds. A preferred salt is the hydrochloride salt.

The term “pharmaceutically acceptable salts” includes derivatives of thedisclosed compounds, wherein the parent compound is modified by makingnon-toxic acid or base addition salts thereof, and further refers topharmaceutically acceptable solvates, including hydrates, of suchcompounds and such salts. Examples of pharmaceutically acceptable saltsinclude, but are not limited to, mineral or organic acid addition saltsof basic residues such as amines; alkali or organic addition salts ofacidic residues such as carboxylic acids; and the like, and combinationscomprising one or more of the foregoing salts. The pharmaceuticallyacceptable salts include non-toxic salts and the quaternary ammoniumsalts of the parent compound formed, for example, from non-toxicinorganic or organic acids. For example, non-toxic acid salts includethose derived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfamic, phosphoric, and nitric; other acceptable inorganicsalts include metal salts such as sodium salt, potassium salt, andcesium salt; and alkaline earth metal salts, such as calcium salt andmagnesium salt; and combinations comprising one or more of the foregoingsalts.

Pharmaceutically acceptable organic salts include salts prepared fromorganic acids such as acetic, trifluoroacetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,HOOC(CH₂)_(n)COOH where n is 0-4; organic amine salts such astriethylamine salt, pyridine salt, picoline salt, ethanolamine salt,triethanolamine salt, dicyclohexylamine salt,N,N′-dibenzylethylenediamine salt; and amino acid salts such asarginate, asparginate, and glutamate, and combinations comprising one ormore of the foregoing salts.

An “effective amount” of a combination of agents (e.g., MEK and RAFinhibitors, or RAF and COT inhibitors, or RAF and an inhibitor targetingMAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL (CrkL), FGR (Fgr), PRKCE (Prkce),PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3)) is an amountsufficient to provide an observable improvement over the baselineclinically observable signs and symptoms of the disorder treated withthe combination.

The pharmaceutical products can be administrated by oral or other forms,e.g., rectally or by parenteral injection. “Oral dosage form” is meantto include a unit dosage form prescribed or intended for oraladministration. An oral dosage form may or may not comprise a pluralityof subunits such as, for example, microcapsules or microtablets,packaged for administration in a single dose.

The pharmaceutical products can be released in various forms.“Releasable form” is meant to include instant release,immediate-release, controlled-release, and sustained-release forms.

“Instant-release” is meant to include a dosage form designed to ensurerapid dissolution of the active agent by modifying the normal crystalform of the active agent to obtain a more rapid dissolution.

“Immediate-release” is meant to include a conventional or non-modifiedrelease form in which greater than or equal to about 50% or morepreferably about 75% of the active agents is released within two hoursof administration, preferably within one hour of administration.

“Sustained-release” or “extended-release” includes the release of activeagents at such a rate that blood (e.g., plasma) levels are maintainedwithin a therapeutic range but below toxic levels for at least about 8hours, preferably at least about 12 hours, more preferably about 24hours after administration at steady-state. The term “steady-state”means that a plasma level for a given active agent or combination ofactive agents, has been achieved and which is maintained with subsequentdoses of the active agent(s) at a level which is at or above the minimumeffective therapeutic level and is below the minimum toxic plasma levelfor a given active agent(s).

The term “treat”, “treated,” “treating” or “treatment” is used herein tomean to relieve, reduce or alleviate at least one symptom of a diseasein a subject. For example, treatment can be diminishment of one orseveral symptoms of a disorder or complete eradication of a disorder,such as cancer. Within the meaning of the present invention, the term“treat” also denote to arrest, delay the onset (i.e., the period priorto clinical manifestation of a disease) and/or reduce the risk ofdeveloping or worsening a disease. The term “protect” is used herein tomean prevent delay or treat, or all, as appropriate, development orcontinuance or aggravation of a disease in a subject. Within the meaningof the present invention, the disease is associated with a cancer.

The term “subject” or “patient” is intended to include animals, whichare capable of suffering from or afflicted with a cancer or any disorderinvolving, directly or indirectly, a cancer. Examples of subjectsinclude mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats,cats, mice, rabbits, rats, and transgenic non-human animals. In certainembodiments, the subject is a human, e.g., a human suffering from, atrisk of suffering from, or potentially capable of suffering fromcancers.

The term “about” or “approximately” usually means within 20%, morepreferably within 10%, and most preferably still within 5% of a givenvalue or range. Alternatively, especially in biological systems, theterm “about” means within about a log (i.e., an order of magnitude)preferably within a factor of two of a given value.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising, “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein.

As specified above, in one aspect, the instant invention provides a drugcombination useful for treating, preventing, arresting, delaying theonset of and/or reducing the risk of developing, or reversing at leastone symptom of cancer, in a subject comprising administering to thesubject a combination therapy, comprising an effective amount of a RAFinhibitor and an effective amount of a MAP3K8 (TPL2/COT) inhibitor, oran effective amount of a RAF inhibitor and an effective amount of MEKinhibitor or an effective amount of a RAF inhibitor and an effectiveamount of a second inhibitor targeting MAP3K8 (TPL2/COT), RAF1 (CRAF),CRKL (CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL(Axl), or PAK3 (Pak3). Preferably, these inhibitors are administered attherapeutically effective dosages which, when combined, provide abeneficial effect. The administration may be simultaneous or sequential.

The term “cancer” is used herein to mean a broad spectrum of tumors,including all solid tumors and hematological malignancies. Examples ofsuch tumors include but are not limited to leukemias, lymphomas,myelomas, carcinomas, metastatic carcinomas, sarcomas, adenomas, nervoussystem cancers and geritourinary cancers. In exemplary embodiments, theforegoing methods are useful in treating adult and pediatric acutelymphoblastic leukemia, acute myeloid leukemia, adrenocorticalcarcinoma, AIDS-related cancers, anal cancer, cancer of the appendix,astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer,bone cancer, osteosarcoma, fibrous histiocytoma, brain cancer, brainstem glioma, cerebellar astrocytoma, malignant glioma, ependymoma,medulloblastoma, supratentorial primitive neuroectodermal tumors,hypothalamic glioma, breast cancer, male breast cancer, bronchialadenomas, Burkitt lymphoma, carcinoid tumor, carcinoma of unknownorigin, central nervous system lymphoma, cerebellar astrocytoma,malignant glioma, cervical cancer, childhood cancers, chroniclymphocytic leukemia, chronic myelogenous leukemia, chronicmyeloproliferative disorders, colorectal cancer, cutaneous T-celllymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewingfamily tumors, extracranial germ cell tumor, extragonadal germ celltumor, extrahepatic bile duct cancer, intraocular melanoma,retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinalstromal tumor, extracranial germ cell tumor, extragonadal germ celltumor, ovarian germ cell tumor, gestational trophoblastic tumor, glioma,hairy cell leukemia, head and neck cancer, hepatocellular cancer,Hodgkin lymphoma, non-Hodgkin lymphoma, hypopharyngeal cancer,hypothalamic and visual pathway glioma, intraocular melanoma, islet celltumors, Kaposi sarcoma, kidney cancer, renal cell cancer, laryngealcancer, lip and oral cavity cancer, small cell lung cancer, non-smallcell lung cancer, primary central nervous system lymphoma, Waldenstrommacroglobulinema, malignant fibrous histiocytoma, medulloblastoma,melanoma, Merkel cell carcinoma, malignant mesothelioma, squamous neckcancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosisfungoides, myelodysplastic syndromes, myeloproliferative disorders,chronic myeloproliferative disorders, nasal cavity and paranasal sinuscancer, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer,ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer,pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorialprimitive neuroectodermal tumors, pituitary cancer, plasma cellneoplasms, pleuropulmonary blastoma, prostate cancer, rectal cancer,rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterinesarcoma, Sezary syndrome, non-melanoma skin cancer, small intestinecancer, squamous cell carcinoma, squamous neck cancer, supratentorialprimitive neuroectodermal tumors, testicular cancer, throat cancer,thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer,trophoblastic tumors, urethral cancer, uterine cancer, uterine sarcoma,vaginal cancer, vulvar cancer, and Wilms tumor.

In particular, the cancer may be associated with a mutation in the B-RAFgene. These cancers include melanoma, breast cancer, colorectal cancers,glioma, lung cancer, ovarian cancer, sarcoma and thyroid cancer.

In a particular embodiment, the therapeutic combination provided hereinis effective for the treatment of moderate to severe cancer in asubject.

Dosages

The optimal dose of the combination of agents for treatment of cancercan be determined empirically for each subject using known methods andwill depend upon a variety of factors, including the activity of theagents; the age, body weight, general health, gender and diet of thesubject; the time and route of administration; and other medications thesubject is taking. Optimal dosages may be established using routinetesting and procedures that are well known in the art.

The amount of combination of agents that may be combined with thecarrier materials to produce a single dosage form will vary dependingupon the individual treated and the particular mode of administration.In some embodiments the unit dosage forms containing the combination ofagents as described herein will contain the amounts of each agent of thecombination that are typically administered when the agents areadministered alone.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound that is the lowest dose effective to producea therapeutic effect. Such an effective dose will generally depend uponthe factors described above and is readily determined by one havingskill in the art.

Generally, therapeutically effective doses of the compounds of thisinvention for a patient, when used for the indicated analgesic effects,will range from about 0.0001 to about 1000 mg per kilogram of bodyweight per day, more preferably from about 0.01 to about 50 mg per kgper day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

Pharmaceutical Formulations and Routes of Administration

Provided herein are pharmaceutical formulations comprising a combinationof agents for the treatment of cancer, e.g., melanoma. Thepharmaceutical formulations may additionally comprise a carrier orexcipient, stabilizer, flavoring agent, and/or coloring agent.

Provided herein are pharmaceutical formulations comprising combinationof agents which can be, for example, a combination of two types ofagents: (1) a RAF inhibitor and/or pharmacologically active metabolites,salts, solvates and racemates of the inhibitor and (2) a MAP3K8(TPL2/COT) inhibitor and/or pharmacologically active metabolites, salts,solvates and racemates of the COT inhibitor. In another embodiment thecombination of agents may be provided for a subject comprising BRAFmutant cells or comprising cells over expressing MAP3K8 (TPL2/COT) andinclude: (1) a RAF inhibitor and/or pharmacologically activemetabolites, salts, solvates and racemates of the inhibitor and (2) aMEK inhibitor and/or pharmacologically active metabolites, salts,solvates and racemates of the MEK inhibitor.

The combination of agents may be administered using a variety of routesof administration known to those skilled in the art. The combination ofagents may be administered to humans and other animals orally,parenterally, sublingually, by aerosolization or inhalation spray,rectally, intracisternally, intravaginally, intraperitoneally, bucally,or topically in dosage unit formulations containing conventionalnontoxic pharmaceutically acceptable carriers, adjuvants, and vehiclesas desired. Topical administration may also involve the use oftransdermal administration such as transdermal patches or ionophoresisdevices. The term parenteral as used herein includes subcutaneousinjections, intravenous, intramuscular, intrasternal injection, orinfusion techniques.

Methods of formulation are well known in the art and are disclosed, forexample, in Remington: The Science and Practice of Pharmacy, MackPublishing Company, Easton, Pa., 19th Edition (1995). Pharmaceuticalcompositions for use in the present invention can be in the form ofsterile, non-pyrogenic liquid solutions or suspensions, coated capsules,suppositories, lyophilized powders, transdermal patches or other formsknown in the art.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3 propanediol or 1,3butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P. and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose any blandfixed oil may be employed including synthetic mono or di glycerides. Inaddition, fatty acids such as oleic acid find use in the preparation ofinjectables. The injectable formulations can be sterilized, for example,by filtration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform may be accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such as polylactidepolyglycolide. Depending upon the ratio of drug to polymer and thenature of the particular polymer employed, the rate of drug release canbe controlled. Examples of other biodegradable polymers includepoly(orthoesters) and poly(anhydrides). Depot injectable formulationsmay also be prepared by entrapping the drug in liposomes ormicroemulsions, which are compatible with body tissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,acetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active compounds, the liquid dosage formsmay contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, EtOAc, benzylalcohol, benzyl benzoate, propylene glycol, 1,3 butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, the oral compositions can alsoinclude adjuvants such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring, and perfuming agents.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulations, ear drops, and the like are also contemplatedas being within the scope of this invention.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Compositions of the invention may also be formulated for delivery as aliquid aerosol or inhalable dry powder. Liquid aerosol formulations maybe nebulized predominantly into particle sizes that can be delivered tothe terminal and respiratory bronchioles.

Aerosolized formulations of the invention may be delivered using anaerosol forming device, such as a jet, vibrating porous plate orultrasonic nebulizer, preferably selected to allow the formation of anaerosol particles having with a mass medium average diameterpredominantly between 1 to 5 m. Further, the formulation preferably hasbalanced osmolarity ionic strength and chloride concentration, and thesmallest aerosolizable volume able to deliver effective dose of thecompounds of the invention to the site of the infection. Additionally,the aerosolized formulation preferably does not impair negatively thefunctionality of the airways and does not cause undesirable sideeffects.

Aerosolization devices suitable for administration of aerosolformulations of the invention include, for example, jet, vibratingporous plate, ultrasonic nebulizers and energized dry powder inhalers,that are able to nebulize the formulation of the invention into aerosolparticle size predominantly in the size range from 1 5 m. Predominantlyin this application means that at least 70% but preferably more than 90%of all generated aerosol particles are within 1 5 m range. A jetnebulizer works by air pressure to break a liquid solution into aerosoldroplets. Vibrating porous plate nebulizers work by using a sonic vacuumproduced by a rapidly vibrating porous plate to extrude a solventdroplet through a porous plate. An ultrasonic nebulizer works by apiezoelectric crystal that shears a liquid into small aerosol droplets.A variety of suitable devices are available, including, for example,AERONEB and AERODOSE vibrating porous plate nebulizers (AeroGen, Inc.,Sunnyvale, Calif.), SIDESTREAM nebulizers (Medic Aid Ltd., West Sussex,England), PARI LC and PARI LC STAR jet nebulizers (Pari RespiratoryEquipment, Inc., Richmond, Va.), and AEROSONIC (DeVilbiss MedizinischeProdukte (Deutschland) GmbH, Heiden, Germany) and ULTRAAIRE (OmronHealthcare, Inc., Vernon Hills, Ill.) ultrasonic nebulizers.

Compounds of the invention may also be formulated for use as topicalpowders and sprays that can contain, in addition to the compounds ofthis invention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the compound in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin. The rate can be controlled by either providing a ratecontrolling membrane or by dispersing the compound in a polymer matrixor gel. The compounds of the present invention can also be administeredin the form of liposomes. As is known in the art, liposomes aregenerally derived from phospholipids or other lipid substances.Liposomes are formed by mono or multi lamellar hydrated liquid crystalsthat are dispersed in an aqueous medium. Any non toxic, physiologicallyacceptable and metabolizable lipid capable of forming liposomes can beused. The present compositions in liposome form can contain, in additionto a compound of the present invention, stabilizers, preservatives,excipients, and the like. The preferred lipids are the phospholipids andphosphatidyl cholines (lecithins), both natural and synthetic. Methodsto form liposomes are known in the art. See, for example, Prescott(ed.), “Methods in Cell Biology,” Volume XIV, Academic Press, New York,1976, p. 33 et seq.

EXAMPLES Example 1 An ORF-Based Functional Screen Identifies SpecificKinases as Drivers of Resistance to B-RAF Inhibition

To identify kinases capable of circumventing RAF inhibition, 597sequence-validated kinase ORF clones representing ˜75% of annotatedkinases (Center for Cancer Systems Biology (CCSB)/Broad Institute KinaseORF Collection) were assembled and stably expressed in A375, aB-RAF^(V600E) malignant melanoma cell line that is sensitive to the RAFkinase inhibitor PLX4720 (Tsai, J. et al. Proc. Natl. Acad. Sci. USA105, 3041-3046 (2008)) (FIG. 1 a, 1 b, Table 3, FIG. 6 c). ORFexpressing cells treated with 1 μM PLX4720 were screened for viabilityrelative to untreated cells and normalized to an assay-specific positivecontrol, MEK1^(S218/222D) (MEK1^(DD)) (Emery, C. M. et al. Proc. Natl.Acad. Sci. USA 106, 20411-20416 (2009).) (Table 4 and summarized in FIG.5). Nine ORFs conferred resistance at levels exceeding two standarddeviations from the mean (FIG. 1 b and Table 4) and were selected forfollow-up analysis (FIG. 7). Three of nine candidate ORFs were receptortyrosine kinases, underscoring the potential of this class of kinases toengage resistance pathways. Resistance effects were validated andprioritized across a multi-point PLX4720 drug concentration scale in theB-RAF^(V600E) cell lines A375 and SKMEL28. The Ser/Thr MAP kinase kinasekinases (MAP3Ks) MAP3K8 (COT/Tpl2) and RAF1 (C-RAF) emerged as topcandidates from both cell lines; these ORFs shifted the PLX4720 GI₅₀ by10-600 fold without affecting viability (Table 5 and FIGS. 8 and 9).CRKL, an ORF that shifted the PLX4720 GI₅₀ to a lesser extent (9.7 foldin SKMEL28 cells; FIG. 8), encodes an adapter protein phosphorylated bytyrosine kinases such as BCR-ABL (Birge, R. B. et al., Cell CommunSignal 7, 13 (2009)), but lacks intrinsic kinase activity. COT and C-RAFreduced sensitivity to PLX4720 in multiple B-RAF^(V600E) cell lines(FIG. 1 c) confirming the ability of these kinases to mediate resistanceto RAF inhibition. A secondary screen in A375 and SKMEL28 prioritizesthe top 9 candidate ORFs across a multipoint PLX4720 concentration scale(FIG. 1 d).

Interestingly, the top two validated kinases are both Ser/Thr MAP kinasekinase kinases (MAP3Ks) known to activate MEK/ERK signaling in severalcontexts. Like B-RAF, C-RAF is a MAP3K in the canonical MAPK cascade(McKay, M. M. and Morrison, D. K. Oncogene 26, 3113-3121 (2007)) thatwas previously implicated in resistance associated with stepwiseselection in vitro using a pan-RAF inhibitor (Montagut, C. et al. CancerRes 68, 4853-4861 (2008)). COT (the protein product of the human MAP3K8gene) is best characterized as the MAP3K (Salmeron, A. et al. EMBO J.15, 817-826 (1996)) downstream of NFKB signaling in inflammatory cells(Banerjee, A. et al., Proc Natl Acad Sci U.S.A. 103, 3274-3279 (2006));however, its functional importance in human cancer has not previouslybeen elucidated.

Example 2 Resistance to B-RAF Inhibition Via MAPK Pathway Activation

Whether the overexpression of these genes was sufficient to activate theMAPK pathway was also tested. At baseline, COT expression increased ERKphosphorylation in a manner comparable to MEK1^(DD), consistent with MAPkinase pathway activation (FIGS. 2 a and 10). Overexpression ofwild-type COT or C-RAF resulted in constitutive phosphorylation of ERKand MEK in the presence of PLX4720, whereas kinase-dead derivatives hadno effect (FIGS. 2 a and 11). Thus, COT and C-RAF drive resistance toRAF inhibition predominantly through re-activation of MAPK signaling.Notably, of the nine candidate ORFs from the initial screen, a subset(3) did not show persistent ERK/MEK phosphorylation following RAFinhibition, suggesting MAPK pathway-independent alteration of drugsensitivity (FIG. 12).

Example 3 C-RAF Activation and Heterodimerization with B-RAF

C-RAF activation and heterodimerization with B-RAF constitute criticalcomponents of the cellular response to B-RAF inhibition. In A375 cells,endogenous C-RAF: B-RAF heterodimers were measurable and induciblefollowing treatment with PLX4720 (FIG. 13). However, endogenous C-RAFphosphorylation at S338—an event required for C-RAF activation—remainedlow (FIG. 13). In contrast, ectopically expressed C-RAF wasphosphorylated on S338 (FIG. 13) and its PLX4720 resistance phenotypewas associated with sustained MEK/ERK activation (FIGS. 2 a and 13).Moreover, ectopic expression of a high-activity C-RAF truncation mutant(C-RAF(W22) was more effective than wild-type C-RAF in mediating PLX4720resistance and ERK activation (FIG. 14), further indicating thatelevated C-RAF activity directs resistance to this agent. Consistentwith this model, oncogenic alleles of NRAS and KRAS conferred PLX4720resistance in A375 cells (FIG. 2 b) and yielded sustained C-RAF(S338)and ERK phosphorylation in the context of drug treatment (FIG. 2 c).Thus, although genetic alterations that engender C-RAF activation (e.g.,oncogenic RAS mutations) tend to show mutual exclusivity withB-RAF^(V600E) mutation, such co-occurring events are favored in thecontext of acquired resistance to B-RAF inhibition.

Example 4 Investigation of COT Expression in Melanoma

While C-RAF has been linked previously to melanoma and MAPK pathwaydependencies (Montagut, C. et al. 2008; Karreth, F. A., DeNicola, G. M.et al., 2009; Dumaz, N. et al. Cancer Res 66, 9483-9491 (2006);Hatzivassiliou, G. et al. Nature (2010); Heidorn, S. J. et al., Cell140, 209-221 (2010); Poulikakos, P. I. et al., Nature (2010)), COT hasnot been described as a melanoma-associated kinase.

The role of COT in melanoma was investigated, and its expression inhuman melanocytes was examined. Primary immortalized melanocytes (B-RAFwild-type) expressed COT (FIG. 2 d), although ectopic B-RAF^(V600E)expression reduced COT mRNA levels (FIG. 15) and rendered COT proteinundetectable (FIG. 2 d). Conversely, whereas ectopically expressed COTwas only weakly detectable in A375 cells (FIGS. 2 a, 2 e),shRNA-mediated depletion of endogenous B-RAF^(V600E) caused an increasein COT protein levels that correlated with the extent of B-RAF knockdown(FIG. 2 e). Moreover, treatment of COT-expressing A375 cells withPLX4720 led to a dose-dependent increase in COT protein (FIG. 2 a)without affecting ectopic COT mRNA levels (FIG. 15). Oncogenic B-RAFantagonizes COT expression largely through altered protein stability(FIGS. 2 a, d, e, and 15), and B-RAF inhibition potentiates theoutgrowth of COT-expressing cells during the course of treatment.Notably, neither C-RAF nor B-RAF alone or in combination was requiredfor ERK phosphorylation in the context of COT expression, even in thepresence of PLX4720 (FIGS. 2 e, 2 f and FIG. 16). As shown, COTexpression is sufficient to induce MAP kinase pathway activation in aRAF-independent manner.

Example 5 COT Expression Predicts Resistance to B-RAF Inhibition inCancer Cell Lines

Whether cell lines expressing elevated COT in a B-RAF^(V600E) backgroundexhibit de novo resistance to PLX4720 treatment was tested. To identifysuch instances, a panel of cell lines was screened for evidence ofMAP3K8/COT copy number gains coincident with the B-RAF^(V600E) mutation.Of 534 cell lines that had undergone copy number analysis and mutationprofiling, 38 cell lines (7.1%) contained the B-RAF^(V600E) mutation.Within this subgroup, two cell lines—OUMS-23 (colon cancer) andRPMI-7951 (melanoma)—also showed evidence of chromosomal copy gainsspanning the MAP3K8/COT locus (FIGS. 3 a and 17) and robust COT proteinexpression (FIGS. 3 b and 18). A panel of melanoma short-term cultureswas also screened for COT protein expression. One of these linesexpressed COT: M307, a short-term culture derived from a B-RAF^(V600E)tumor that developed resistance to allosteric MEK inhibition followinginitial disease stabilization (FIG. 3 c). All three cell lines wererefractory to PLX4720 treatment, exhibiting GI₅₀ values in the range of8-10 μM (FIG. 3 d) and showing sustained ERK phosphorylation in thecontext of B-RAF inhibition (FIGS. 3 e, 3 f). OUMS-23 and RPMI-7951 areMAPK pathway inhibitor-naïve cell lines; thus, these results demonstratethat COT confers de novo resistance to RAF inhibition (a phenomenonobserved in ˜10% of B-RAF^(V600E) melanomas).

Example 6 COT Expression in Patients Treated with a RAF Inhibitor

COT expression in the context of resistance to the clinical RAFinhibitor PLX4032 was examined by obtaining biopsy material from 3patients with metastatic, B-RAF^(V600E) melanoma. Each case consisted offrozen, lesion-matched biopsy material obtained prior to and duringtreatment (“pre-treatment” and “on-treatment”; FIG. 3 g, Table 6);additionally, one sample contained two independent biopsy specimens fromthe same relapsing tumor site (“post-relapse”; FIG. 3 g). Consistentwith the experimental models presented above, quantitative real-timeRT-PCR (qRT/PCR) analysis revealed increased COT mRNA expressionconcurrent with PLX4032 treatment in 2 of 3 cases. COT mRNA levels werefurther increased in a relapsing specimen relative to its pre-treatmentand on-treatment counterparts (FIG. 3 g, Patient #1). An additional,unmatched relapsed malignant melanoma biopsy showed elevated COT mRNAexpression comparable to levels observed in RAF inhibitor-resistant,COT-amplified cell lines (FIG. 19). This specimen also exhibited robustMAPK pathway activation and elevated expression of B-RAF, C-RAF and COTrelative to matched normal skin or B-RAF^(V600E) cell lines (FIG. 19).Sequencing studies of this tumor revealed no additional mutations inBRAF, NRAS or KRAS (data not shown). These analyses provided clinicalevidence that COT-dependent mechanisms are operant in PLX4032-resistantmalignant melanomas.

Example 7 COT Regulation of MEK/ERK Phosphorylation

Whether COT actively regulates MEK/ERK phosphorylation in B-RAF^(V600E)cells that harbor naturally elevated COT expression was tested byintroducing shRNA constructs targeting COT into RPMI-7951 cells.Depletion of COT suppressed RPMI-7951 viability (FIG. 20) and decreasedERK phosphorylation (FIG. 3 h); thus, targeting COT kinase activitysuppresses MEK/ERK phosphorylation in cancer cells with COToverexpression or amplification. Additionally, the targeting COT kinaseactivity in the presence of a B-RAF inhibitor (PLX4720) suppressesMEK/IRK phosphorylation (FIG. 3 h). Treatment of RPMI-7951 cells with asmall molecule COT kinase inhibitor (Wyeth, Abbot compound ID 9549300)(George, D. et al., Bioorg. Med. Chem. Lett. 18, 4952-4955 (2008);Hirata, K. et al., Biol. Pharm. Bull. 33, 133-137 (2010); Lee, K. M. etal., Cancer Res. 69, 8043-8049 (2009)) resulted in dose-dependentsuppression of MEK and ERK phosphorylation, providing additionalevidence that COT contributes to MEK/ERK activation in these cells (FIG.3 i).

Example 8 COT-Expressing B-RAF^(V600E) Cell Lines Exhibit Resistance toAllosteric MEK Inhibitors

Whether COT-expressing cancer cells remain sensitive to MAPK pathwayinhibition at a target downstream of COT or RAF was analyzed. TheOUMS-23 and RPMI-7951 cell lines were queried for sensitivity to theMEK1/2 inhibitor CI-1040. Both cell lines were refractory to MEKinhibition (FIG. 4 a) and displayed sustained ERK phosphorylation evenat 1 μM CI-1040 (FIG. 4 b). Ectopic COT expression in A375 and SKMEL28cells also conferred decreased sensitivity to the MEK inhibitors CI-1040and AZD6244, suggesting that COT expression alone was sufficient toinduce this phenotype (FIGS. 4 c, 4 d and 21). Similar to resultsobserved with pharmacological MEK inhibitors, MEK1/2 knockdown onlymodestly suppressed COT-mediated ERK phosphorylation in A375 cells (FIG.22). These data demonstrate that COT activates ERK throughMEK-independent and MEK-dependent mechanisms. Furthermore, an in vitrokinase assay using recombinant COT and ERK1 was performed, and it wasdemonstrated that recombinant COT induced pThr202/Tyr204 phosphorylationof ERK1 in vitro (FIG. 22). Thus, COT expression potentiates ERKactivation in a MEK-independent manner.

Example 9 Combinatorial MAPK Pathway Inhibition to Suppress CellProliferation

The use of RAF and MEK inhibitors in combination can override resistanceto single-agents as shown in FIG. 23. It was tested whether the combinedRAF/MEK inhibition might circumvent COT-driven resistance. In thesetting of ectopic COT expression, exposure to AZD6244 or CI-1040 incombination with PLX470 (1 μM each) reduced cell growth and pERKexpression more effectively than did single-agent PLX4720, even atconcentrations of 10 μM (FIGS. 4 e, 4 f and 23). These data underscorethe importance of this pathway in B-RAF^(V600E) tumor cells anddemonstrate that dual B-RAF/MEK inhibition helps circumvent resistanceto RAF inhibitors.

Methods

Center for Cancer Systems Biology (CCSB)/Broad Institute Kinase OpenReading Frame Collection

A library of 597 kinase ORFs in pDONR-223 Entry vectors (Invitrogen) wasassembled. Individual clones were end-sequenced using vector-specificprimers in both directions. Clones with substantial deviations fromreported sequences were discarded. Entry clones and sequences areavailable via Addgene (http://www.addgene.org/human_kinases). KinaseORFs were assembled from multiple sources; 337 kinases were isolated assingle clones from the ORFeome 5.1 collection(http://horfdb.dfci.harvard.edu), 183 kinases were cloned from normalhuman tissue RNA (Ambion) by reverse transcription and subsequent PCRamplification to add Gateway sequences (Invitrogen), 64 kinases werecloned from templates provided by the Harvard Institute of Proteomics(HIP), and 13 kinases were cloned into the Gateway system from templatesobtained from collaborating laboratories. The Gateway-compatiblelentiviral vector pLX-Blast-V5 was created from the pLKO.1 backbone. LRClonase enzymatic recombination reactions were performed to introducethe 597 kinases into pLX-Blast-V5 according to the manufacturer'sprotocol (Invitrogen).

High Throughput ORF Screening

A375 melanoma cells were plated in 384-well microtiter plates (500 cellsper well). The following day, cells were spin-infected with thelentivirally-packaged kinase ORF library in the presence of 8 ug/mlpolybrene. 48 hours post-infection, media was replaced with standardgrowth media (2 replicates), media containing 1 μM PLX4720 (2replicates, 2 time points) or media containing 10 ug/ml blasticidin (2replicates). After four days and 6 days, cell growth was assayed usingCell Titer-Glo (Promega) according to manufacturer instructions. Theentire experiment was performed twice.

Identification of Candidate Resistance ORFs

Raw luminescence values were imported into Microsoft Excel. Infectionefficiency was determined by the percentage of duplicate-averaged rawluminescence in blasticidin selected cells relative to non-selectedcells. ORFs with an infection efficiency of less than 0.70 were excludedfrom further analysis along with any ORF having a standard deviationof >15,000 raw luminescence units between duplicates. To identify ORFswhose expression affects proliferation, we compared theduplicate-averaged raw luminescence of individual ORFs against theaverage and standard deviation of all control-treated cells via thez-score, or standard score, below,

$Z = \frac{\chi - \mu}{\sigma}$

where x=average raw luminescence of a given ORF, μ=the mean rawluminescence of all ORFs and σ=the standard deviation of the rawluminescence of all wells. Any individual ORF with a z-score >+2 or <−2was annotated as affecting proliferation and removed from finalanalysis. Differential proliferation was determined by the percentage ofduplicate-averaged raw luminescence values in PLX4720 (1 μM) treatedcells relative to untreated cells. Subsequently, differentialproliferation was normalized to the positive control for PLX4720resistance, MEK1^(s218/222D) (MEK1^(DD)), with MEK1^(DD) differentialproliferation=1.0. MEK1^(DD) normalized differential proliferation foreach individual ORF was averaged across two duplicate experiments, withtwo time points for each experiment (day 4 and day 6). A z-score wasthen generated, as described above for average MEK1^(DD) normalizeddifferential proliferation. ORFs with a z-score of >2 were consideredhits and were followed up in the secondary screen.

ORF and shRNA Expression

ORFs were expressed from pLX-Blast-V5 (lentiviral) or pWZL-Blast,pBABE-Puro or pBABE-zeocin (retroviral) expression plasmids. Forlentiviral transduction, 293T cells were transfected with 1 μg ofpLX-Blast-V5-ORF or pLKO.1-shRNA, 900 ng Δ8.9 (gag, pol) and 100 ngVSV-G using 6 μl Fugene6 transfection reagent (Roche). Viral supernatantwas harvested 72 h post-transfection. Mammalian cells were infected at a1:10-1:20 dilution of virus in 6-well plates in the presence of 5 μg/mlpolybrene and centrifuged at 2250 RPM for 1 h at 37° C. Twenty-fourhours after infection blasticidin (pLX-Blast-V5, 10 μg/ml) or puro(pLKO.1, 0.75 μg/ml) was added and cells were selected for 48 hrs. Forretrovirus production, 293T were transfected with 1 μg of retroviralplasmid-ORF, 1 μg pCL-AMPHO and 100 ng VSV-G, as described above. Cellswere infected with retrovirus containing supernatant at a 1:2 dilutionin 5 μg/ml polybrene overnight, followed by media change to growthmedium. Infection was repeated once more (twice total), followed byselection, above.

Secondary Screen

A375 (1.5×10³) and SKMEL28 cells (3×10³) were seeded in 96-well platesfor 18 h. ORF-expressing lentivirus was added at a 1:10 dilution in thepresence of 8 μg/ml polybrene, and centrifuged at 2250 RPM and 37° C.for 1 h. Following centrifugation, virus-containing media was changed tonormal growth media and allowed to incubate for 18 h. Twenty-four hoursafter infection, DMSO (1:1000) or 10×PLX4720 (in DMSO) was added to afinal concentration of 100, 10, 1, 0.1, 0.01, 0.001, 0.0001 or 0.00001μM. Cell viability was assayed using WST-1 (Roche), per manufacturerrecommendation, 4 days after the addition of PLX4720.

Cell Lines and Reagents

A375, SKMEL28, SKMEL30, COLO-679, WM451lu, SKMEL5, Malme 3M, SKMEL30,WM3627, WM1976, WM3163, WM3130, WM3629, WM3453, WM3682 and WM3702 wereall grown in RPMI (Cellgro), 10% FBS and 1% penicillin/streptomycin.M307 was grown in RPMI (Cellgro), 10% FBS and 1% penicillin/streptomycinsupplemented with 1 mM sodium pyruvate. 293T and OUMS-23 were grown inDMEM (Cellgro), 10% FBS and 1% penicillin/streptomycin. RPMI-7951 cells(ATCC) were grown in MEM (Cellgro), 10% FBS and 1%penicillin/streptomycin. Wild-type primary melanocytes were grown inHAM's F10 (Cellgro), 10% FBS and 1% penicillin/streptomycin.B-RAF^(V600E)-expressing primary melanocytes were grown in TIVA media[Ham's F-10 (Cellgro), 7% FBS, 1% penicillin/streptomycin, 2 mMglutamine (Cellgro), 100 uM IBMX, 50 ng/ml TPA, 1 mM dbcAMP (Sigma) and1 μM sodium vanadate]. CI-1040 (PubChem ID: 6918454) was purchased fromShanghai Lechen International Trading Co., AZD6244 (PubChem ID:10127622) from Selleck Chemicals, and PLX4720 (PubChem ID: 24180719)from Symansis. RAF265 (PubChem ID: 11656518) was a generous gift fromNovartis Pharma AG. Unless otherwise indicated, all drug treatments werefor 16 h. Activated alleles of NRAS and KRAS have been previouslydescribed. (Boehm, J. S. et al. Cell 129, 1065-1079 (2007); Lundberg, A.S. et al. Oncogene 21, 4577-4586 (2002)).

Pharmacologic Growth Inhibition Assays

Cultured cells were seeded into 96-well plates (3,000 cells per well)for all melanoma cell lines; 1,500 cells were seeded for A375.Twenty-four hours after seeding, serial dilutions of the relevantcompound were prepared in DMSO added to cells, yielding final drugconcentrations ranging from 100 μM to 1×105 μM, with the final volume ofDMSO not exceeding 1%. Cells were incubated for 96 h following additionof drug. Cell viability was measured using the WST1 viability assay(Roche). Viability was calculated as a percentage of control (untreatedcells) after background subtraction. A minimum of six replicates wereperformed for each cell line and drug combination. Data fromgrowth-inhibition assays were modeled using a nonlinear regression curvefit with a sigmoid dose-response. These curves were displayed and GI50generated using GraphPad Prism 5 for Windows (GraphPad).Sigmoid-response curves that crossed the 50% inhibition point at orabove 10 μM have GI50 values annotated as >10 μM. For single-dosestudies, the identical protocol was followed, using a single dose ofindicated drug (1 μM unless otherwise noted).

Immunoblots and Immunoprecipitations

Cells were washed twice with ice-cold PBS and lysed with 1% NP-40 buffer[150 mM NaCl, 50 mM Tris pH 7.5, 2 mM EDTA pH 8, 25 mM NaF and 1% NP-40]containing 2× protease inhibitors (Roche) and 1× Phosphatase InhibitorCocktails I and II (CalBioChem). Lysates were quantified (Bradfordassay), normalized, reduced, denatured (95° C.) and resolved by SDS gelelectrophoresis on 10% Tris/Glycine gels (Invitrogen). Protein wastransferred to PVDF membranes and probed with primary antibodiesrecognizing pERK1/2 (T202/Y204), pMEK1/2 (S217/221), MEK1/2, MEK1, MEK2,C-RAF (rabbit host), pC-RAF (pS338) (Cell Signaling Technology;1:1,000), V5-HRP (Invitrogen; (1:5,000), COT (1:500), B-RAF (1:2,000),Actin (1:1,000), Actin-HRP (1:1,000; Santa Cruz)), C-RAF (mouse host;1:1,000; BD Transduction Labs), Vinculin (Sigma; 1:20,000), AXL (1:500;R&D Systems). After incubation with the appropriate secondary antibody(anti-rabbit, anti-mouse IgG, HRP-linked; 1:1,000 dilution, CellSignaling Technology or anti-goat IgG, HRP-linked; 1:1,000 dilution;Santa Cruz), proteins were detected using chemiluminescence (Pierce).Immunoprecipitations were performed overnight at 4° C. in 1% NP-40 lysisbuffer, as described above, at a concentration of 1 μg/μl total proteinusing an antibody recognizing C-RAF (1:50; Cell Signaling Technology).Antibody: antigen complexes were bound to Protein A agarose (25 μL, 50%slurry; Pierce) for 2 hrs. at 4° C. Beads were centrifuged and washedthree times in lysis buffer and eluted and denatured (95° C.) in 2×reduced sample buffer (Invitrogen). Immunoblots were performed as above.Phospho-protein quantification was performed using NIH Image J.

Lysates from tumor and matched normal skin were generated by mechanicalhomogenization of tissue in RIPA [50 mM Tris (pH 7.4), 150 mM NaCl, 1 mMEDTA, 0.1% SDS, 1.0% NaDOC, 1.0% Triton X-100, 25 mM NaF, 1 mM NA3VO4]containing protease and phosphatase inhibitors, as above. Subsequentnormalization and immunoblots were performed as above.

Biopsied Melanoma Tumour Material

Biopsied tumor material consisted of discarded and de-identified tissuethat was obtained with informed consent and characterized under protocol02-017 (paired samples, Massachusetts General Hospital) and 07-087(unpaired sample, Dana-Farber Cancer Institute). For paired specimens,‘on-treatment’ samples were collected 10-14 days after initiation ofPLX4032 treatment (Table 6).

Inhibition of COT Kinase Activity

Adherent RPMI-7951 cells were washed twice with 1×PBS and incubatedovernight in serum-free growth media. Subsequently,4-(3-Chloro-4-fluorophenylamino)-6-(pyridin-3-yl-methylamino)-3-cyano-[1,7]-naphthyridine(EMD; TPL2 inhibitor I; Cat#: 616373, PubChem ID: 9549300), suspended inDMSO at the indicated concentration, was added to cells for 1 hour,after which protein extracts were made as described above.

Quantitative RT-PCR

mRNA was extracted from cell lines and fresh-frozen tumors using theRNeasy kit (Qiagen). Total mRNA was used for subsequent reversetranscription using the SuperScript III First-Strand Synthesis SuperMix(Invitrogen) for cell lines and unpaired tumor samples, and theSuperScript VILO cDNA synthesis kit (Invitrogen) for paired frozen tumorsamples. 5 μl of the RT reaction was used for quantitative PCR usingSYBR Green PCR Master Mix and gene-specific primers, in triplicate,using an ABI 7300 Real Time PCR System. Primers used for detection areas follows:

Primer Sequence SEQ.ID. NO. COT forward CAAGTGAAGAGCCAGCAGTTTSEQ. ID.NO: 1 COT reverse GCAAGCAAATCCTCCACAGTTC SEQ. ID.NO: 2TBP forward CCCGAAACGCCGAATATAATCC SEQ. ID.NO: 3 TBP reverseGACTGTTCTTCACTCTTGGCTC SEQ. ID.NO: 4 GAPDH  CATCATCTCTGCCCCCTCTSEQ. ID.NO: 5 forward GAPDH  GGTGCTAAGCAGTTGGTGGT SEQ. ID.NO: 6 reverse

In Vitro Kinase Assay

In vitro kinase assays were performed as previously described using 1 μgeach of COT (amino acids 30-397, R&D Systems) and inactive ERK1(Millipore).

Cellular Viability Assays

Adherent RPMI-7951 cells were infected with virus expressing shRNAsagainst COT or Luciferase as described above. Following selection, cellswere plated (1.5×10⁵ cells/well) onto a 24-well plate in quadruplicate.Viable cells were counted via trypan blue exclusion using a VI-CELL CellViability Analyzer, per manufacturer's specifications. Quadruplicatecell counts were averaged and normalized relative to that of the controlshRNA.

The Cancer Cell Line Encyclopedia (CCLE)

The Cancer Cell Line Encyclopedia (COLE) project is a collaborationbetween the Broad Institute, the Novartis Institutes for BiomedicalResearch (NIBR) and the Genomics Institute of the Novartis ResearchFoundation (GNF) to conduct a detailed genetic and pharmacologiccharacterization of a large panel of human cancer models, to developintegrated computational analyses that link distinct pharmacologicvulnerabilities to genomic patterns and to translate cell lineintegrative genomics into cancer patient stratification. Chromosomalcopy number and gene expression data used for this study are availableonline at http://www.broadinstitute.org/cgi-bin/cancer/datasets.cgi.

Expression Profiling of Cancer Cell Lines

Oligonucleotide microarray analysis was carried out using the GeneChipHuman Genome U133 Plus 2.0 Affymetrix expression array (Affymetrix).Samples were converted to labeled, fragmented, cRNA following theAffymetrix protocol for use on the expression microarray.

shRNA Constructs Used (pLKO.1)

The DNA sequences for preparing the shRNA constructs used were asfollows:

TRC SEQ. ID. shRNA Identifier NM No. Sequence NO. shLuc TRCN NACTTCGAAATGTCCGTTCGGTT SEQ. ID. 0000072243 NO. 7 shBRAF(1) TRCNNM_004333.2- CTTCGAAATGTCCGTTCGGTT SEQ. ID. 0000006289 1106s1c1 NO. 8shBRAF(2) TRCN NM_004333.2- GCTGGTTTCCAAACAGAGGAT SEQ. ID. 00000062912267s1c1 NO. 9 shCRAF(1) TRCN NM_002880.x- CGGAGATGTTGCAGTAAAGATSEQ. ID. 0000001066 1236s1c1 NO. 10 shCRAF(2) TRCN NM_002880.x-GAGACATGAAATCCAACAATA SEQ. ID. 0000001068 1529s1c1 NO. 11 shMEK1(1) TRCNNM_002755.x- GATTACATAGTCAACGAGCCT SEQ. ID. 0000002332 1015s1c1 NO. 12shMEK1(2) TRCN NM_002755.x- GCTTCTATGGTGCGTTCTACA SEQ. ID. 0000002329455s1c1 NO. 13 shMEK2(1) TRCN NM_030662.2- TGGACTATATTGTGAACGAGCSEQ. ID. 0000007007 1219s1c1 NO. 14 shMEK2(2) TRCN NM_030662.2-CCAACATCCTCGTGAACTCTA SEQ. ID. 0000007005 847s1c1 NO. 15 shCOT(1) TRCNNM_005204.x- CAAGAGCCGCAGACCTACTAA SEQ. ID. 0000010013 1826s1c1 NO. 16shCOT(2) TRCN NM_005204.2- GATGAGAATGTGACCTTTAAG SEQ. ID. 00001965182809s1c1 NO. 17

The definitions and disclosures provided herein govern and supersede allothers incorporated by reference. Although the invention herein has beendescribed in connection with preferred embodiments thereof, it will beappreciated by those skilled in the art that additions, modifications,substitutions, and deletions not specifically described may be madewithout departing from the spirit and scope of the invention as definedin the appended claims. It is therefore intended that the foregoingdetailed description be regarded as illustrative rather than limiting,and that it be understood that it is the following claims, including allequivalents, that are intended to define the spirit and scope of thisinvention.

TABLE 3 CCSB/Broad Institute Kinase ORF Library description and ORFclassification hGENE GENE ID DESCRIPTION KINASE CLASS AAK1 22848 AP2associated kinase 1 protein kinase (NRS/TK) ABL1 25 v-abl Abelson murineleukemia viral oncogene homolog 1 protein kinase (NRTK) ABL2 27 v-ablAbelson murine leukemia viral oncogene homolog 2 (arg, protein kinase(NRTK) Abelson-related gene) ACVR1 90 activin A receptor, type I proteinkinase (RS/TK) ACVR1B 91 activin A receptor, type IB protein kinase(RS/TK) ACVR1C 130399 activin A receptor, type IC protein kinase (RS/TK)ACVR2A 92 activin A receptor, type II protein kinase (RS/TK) ACVR2B 93activin A receptor, type IIB protein kinase (RS/TK) ACVRL1 94 activin Areceptor type II-like 1 protein kinase (RS/TK) ADCK1 57143 aarF domaincontaining kinase 1 protein kinase ADCK2 90956 aarF domain containingkinase 2 protein kinase ADCK4 79934 aarF domain containing kinase 4protein kinase ADPGK 83440 ADP-dependent glucokinase kinase relatedADRBK1 156 adrenergic, beta, receptor kinase 1 protein kinase (NRS/TK)ADRBK2 157 adrenergic, beta, receptor kinase 2 protein kinase (NRS/TK)AGK 55750 multiple substrate lipid kinase; MULK kinase related AK1 203adenylate kinase 1 nucleotide kinase AK2 204 adenylate kinase 2nucleotide kinase AK3 205 adenylate kinase 3 nucleotide kinase AK3L150808 adenylate kinase 3 like 1 nucleotide kinase AK7 122481 adenylatekinase 7 nucleotide kinase AKT1 207 v-akt murine thymoma viral oncogenehomolog 1 protein kinase (NRS/TK) AKT3 10000 v-akt murine thymoma viraloncogene homolog 3 (protein kinase protein kinase B, gamma) (NRS/TK)ALDH18A1 5832 aldehyde dehydrogenase 18 family, member A1; ALDH18A1kinase related ALK 238 anaplastic lymphoma kinase (Ki-1) protein kinase(RTK) ALPK1 80216 alpha-kinase 1 protein kinase (NRS/TK) ALPK2 115701alpha-kinase 2 protein kinase (NRS/TK) ALS2CR7 65061 amyotrophic lateralsclerosis 2 (juvenile) chromosome region, protein kinase candidate 7(NRS/TK) AMHR2 269 anti-Mullerian hormone receptor, type II proteinkinase (RS/TK) ARAF 369 v-raf murine sarcoma 3611 viral oncogene homolog1 protein kinase (NRS/TK) ARSG 22901 arylsulfatase G; ARSG kinaserelated ASCIZ 23300 ATM/ATR-Substrate Chk2-Interacting Zn2+-fingerprotein; ASCIZ protein kinase (NRS/TK) AURKA 6790 serine/threoninekinase 6 protein kinase (NRS/TK) AURKB 9212 aurora kinase B proteinkinase (NRS/TK) AURKC 6795 aurora kinase C protein kinase (NRS/TK) AXL558 AXL receptor tyrosine kinase protein kinase (RTK) BCKDK 10295branched chain alpha-ketoacid dehydrogenase kinase protein kinase BLK640 B lymphoid tyrosine kinase protein kinase (NRTK) BMP2K 55589 BMP2inducible kinase protein kinase (NRS/TK) BMP2KL 347359 BMP2 induciblekinase-like protein kinase (NRS/TK) BMPR1A 657 bone morphogeneticprotein receptor, type IA protein kinase (RS/TK) BMPR1B 658 bonemorphogenetic protein receptor, type IB protein kinase (RS/TK) BMPR2 659bone morphogenetic protein receptor, type II (serine/threonine proteinkinase (RS/TK) kinase) BMX 660 BMX non-receptor tyrosine kinase proteinkinase (NRTK) BRAF 673 v-raf murine sarcoma viral oncogene homolog B1protein kinase (NRS/TK) BRD3 8019 bromodomain containing 3 proteinkinase (NRS/TK) BRD4 23476 bromodomain containing 4 protein kinase(NRS/TK) BRSK1 84446 KIAA1811 protein protein kinase (NRS/TK) BRSK2 9024serine/threonine kinase 29 protein kinase (NRS/TK) BTK 695 Brutonagammaglobulinemia tyrosine kinase protein kinase (NRTK) BUB1 699 BUB1budding uninhibited by benzimidazoles 1 homolog (yeast) protein kinase(NRS/TK) BUB1B 701 BUB1 budding uninhibited by benzimidazoles 1 homologbeta protein kinase (yeast) (NRS/TK) C1orf57 84284 chromosome 1 openreading frame 57; C1orf57 kinase related C9orf95 54981 chromosome 9 openreading frame 95; C9orf95 kinase related C9orf98 158067 chromosome 9open reading frame 98; C9orf98 nucleotide kinase CABC1 56997 chaperone,ABC1 activity of bc1 complex like (S. pombe) protein kinase CALM1 801calmodulin 1 (phosphorylase kinase, delta) kinase related CALM2 805calmodulin 2 (phosphorylase kinase, delta) kinase related CALM3 808calmodulin 3 (phosphorylase kinase, delta) kinase related CAMK1 8536calcium/calmodulin-dependent protein kinase I protein kinase (NRS/TK)CAMK1D 57118 calcium/calmodulin-dependent protein kinase ID proteinkinase (NRS/TK) CAMK1G 57172 calcium/calmodulin-dependent protein kinaseIG protein kinase (NRS/TK) CAMK2A 815 calcium/calmodulin-dependentprotein kinase (CaM kinase) II protein kinase alpha (NRS/TK) CAMK2B 816calcium/calmodulin-dependent protein kinase (CaM kinase) II proteinkinase beta (NRS/TK) CAMK2D 817 calcium/calmodulin-dependent proteinkinase (CaM kinase) II protein kinase delta (NRS/TK) CAMK2G 818calcium/calmodulin-dependent protein kinase (CaM kinase) II proteinkinase gamma (NRS/TK) CAMK4 814 calcium/calmodulin-dependent proteinkinase IV protein kinase (NRS/TK) CAMKK1 84254calcium/calmodulin-dependent protein kinase kinase 1, alpha proteinkinase (NRS/TK) CAMKK2 10645 calcium/calmodulin-dependent protein kinasekinase 2, beta protein kinase (NRS/TK) CAMKV 79012 hypothetical proteinMGC8407 protein kinase (NRS/TK) CARD11 84433 caspase recruitment domainfamily, member 11; CARD11 nucleotide kinase CARKL 23729 carbohydratekinase-like carbohydrate kinase CASK 8573 calcium/calmodulin-dependentserine protein kinase (MAGUK nucleotide kinase family) CCL2 6347chemokine (C-C motif) ligand 2; CCL2 protein kinase CCL4 6351 chemokine(C-C motif) ligand 4; CCL4 protein kinase (RTK) CCRK 23552 cell cyclerelated kinase protein kinase (NRS/TK) CD2 914 CD2 antigen (p50), sheepred blood cell receptor; CD2 protein kinase CDC2 983 cell division cycle2, G1 to S and G2 to M protein kinase (NRS/TK) CDC2L1 984 cell divisioncycle 2-like 1 (PITSLRE proteins) protein kinase (NRS/TK) CDC2L2 985cell division cycle 2-like 2 (PITSLRE proteins) protein kinase (NRS/TK)CDC2L6 23097 cell division cycle 2-like 6 (CDK8-like) protein kinase(NRS/TK) CDC42BPG 55561 CDC42 binding protein kinase gamma (DMPK-like)protein kinase (NRS/TK) CDC7 8317 CDC7 cell division cycle 7 (S.cerevisiae) protein kinase (NRS/TK) CDK10 8558 cyclin-dependent kinase(CDC2-like) 10 protein kinase (NRS/TK) CDK2 1017 cyclin-dependent kinase2 protein kinase (NRS/TK) CDK3 1018 cyclin-dependent kinase 3 proteinkinase (NRS/TK) CDK4 1019 cyclin-dependent kinase 4 protein kinase(NRS/TK) CDK5 1020 cyclin-dependent kinase 5 protein kinase (NRS/TK)CDK5R1 8851 cyclin-dependent kinase 5, regulatory subunit 1 (p35)protein kinase (NRS/TK) CDK6 1021 cyclin-dependent kinase 6 proteinkinase (NRS/TK) CDK7 1022 cyclin-dependent kinase 7 (MO15 homolog,Xenopus laevis, cdk- protein kinase activating kinase) (NRS/TK) CDK81024 cyclin-dependent kinase 8 protein kinase (NRS/TK) CDK9 1025cyclin-dependent kinase 9 (CDC2-related kinase) protein kinase (NRS/TK)CDKL1 8814 cyclin-dependent kinase-like 1 (CDC2-related kinase) proteinkinase (NRS/TK) CDKL2 8999 cyclin-dependent kinase-like 2 (CDC2-relatedkinase) protein kinase (NRS/TK) CDKL3 51265 cyclin-dependent kinase-like3 protein kinase (NRS/TK) CDKL4 344387 cyclin-dependent kinase-like 4protein kinase (NRS/TK) CDKL5 6792 cyclin-dependent kinase-like 5protein kinase (NRS/TK) CHEK1 1111 CHK1 checkpoint homolog (S. pombe)protein kinase (NRS/TK) CHEK2 11200 CHK2 checkpoint homolog (S. pombe)protein kinase (NRS/TK) CHKA 1119 choline kinase alpha kinase relatedCIB1 10519 calcium and integrin binding 1 (calmyrin); CIB1 kinaserelated CIB4 130106 calcium and integrin binding family member 4; CIB4kinase related CKB 1152 creatine kinase, brain kinase related CKM 1158creatine kinase, muscle kinase related CKMT1A 548596 creatine kinase,mitochondrial 1A; CKMT1A kinase related CKMT2 1160 creatine kinase,mitochondrial 2 (sarcomeric) kinase related CKS1B 1163 CDC28 proteinkinase regulatory subunit 1B protein kinase CKS2 1164 CDC28 proteinkinase regulatory subunit 2 protein kinase CLK1 1195 CDC-like kinase 1protein kinase (NRS/TK) CLK2 1196 CDC-like kinase 2 protein kinase(NRS/TK) CLK3 1198 CDC-like kinase 3 protein kinase (NRS/TK) COASY 80347Coenzyme A synthase; COASY kinase related COL4A3BP 10087 collagen, typeIV, alpha 3 (Goodpasture antigen) binding protein kinase protein;COL4A3BP CRKL 1399 v-crk sarcoma virus CT10 oncogene homolog(avian)-like; CRKL kinase related CSF1R 1436 colony stimulating factor 1receptor, formerly McDonough feline protein kinase (RTK) sarcoma viral(v-fms) oncogene homolog CSK 1445 c-src tyrosine kinase protein kinase(NRTK) CSNK1A1 1452 casein kinase 1, alpha 1 protein kinase (NRS/TK)CSNK1A1L 122011 casein kinase 1, alpha 1-like protein kinase (NRS/TK)CSNK1D 1453 casein kinase 1, delta protein kinase (NRS/TK) CSNK1E 1454casein kinase 1, epsilon protein kinase (NRS/TK) CSNK1G1 53944 caseinkinase 1, gamma 1 protein kinase (NRS/TK) CSNK1G2 1455 casein kinase 1,gamma 2 protein kinase (NRS/TK) CSNK1G3 1456 casein kinase 1, gamma 3protein kinase (NRS/TK) CSNK2A1 1457 casein kinase 2, alpha 1polypeptide protein kinase (NRS/TK) CSNK2B 1460 casein kinase 2, betapolypeptide protein kinase (NRS/TK) DAK 26007 dihydroxyacetone kinase 2homolog (S. cerevisiae); DAK kinase related DAPK1 1612 death-associatedprotein kinase 1 protein kinase (NRS/TK) DAPK2 23604 death-associatedprotein kinase 2 protein kinase (NRS/TK) DAPK3 1613 death-associatedprotein kinase 3 protein kinase (NRS/TK) DCAKD 79877 dephospho-CoAkinase domain containing; DCAKD kinase related DCAMKL2 166614hypothetical protein MGC45428 protein kinase (NRS/TK) DCK 1633deoxycytidine kinase nucleotide kinase DDR1 780 discoidin domainreceptor family, member 1 protein kinase (RTK) DDR2 4921 discoidindomain receptor family, member 2 protein kinase (RTK) DGKA 1606diacylglycerol kinase, alpha 80 kDa kinase related DGKB 1607diacylglycerol kinase, beta 90 kDa kinase related DGKG 1608diacylglycerol kinase, gamma 90 kDa kinase related DGKK 139189diacylglycerol kinase, kappa; DGKK kinase related DGKZ 8525diacylglycerol kinase, zeta 104 kDa kinase related DGUOK 1716deoxyguanosine kinase nucleotide kinase DKFZp434B1231 91156 eEF1A2binding protein; DKFZp434B1231 protein kinase (NRS/TK) DKFZp761P0423157285 hypothetical protein DKFZp761P0423 protein kinase (RS/TK) DLG11739 discs, large homolog 1 (Drosophila); DLG1 nucleotide kinase DLG31741 discs, large homolog 3 (neuroendocrine-dlg, Drosophila); DLG3nucleotide kinase DTYMK 1841 deoxythymidylate kinase (thymidylatekinase) nucleotide kinase DYRK1A 1859 dual-specificitytyrosine-(Y)-phosphorylation regulated kinase 1A protein kinase (NRS/TK)DYRK1B 9149 dual-specificity tyrosine-(Y)-phosphorylation regulatedkinase 1B protein kinase (NRS/TK) DYRK2 8445 dual-specificitytyrosine-(Y)-phosphorylation regulated kinase 2 protein kinase (NRS/TK)DYRK3 8444 dual-specificity tyrosine-(Y)-phosphorylation regulatedkinase 3 protein kinase (NRS/TK) DYRK4 8798 dual-specificitytyrosine-(Y)-phosphorylation regulated kinase 4 protein kinase (NRS/TK)EEF2K 29904 eukaryotic elongation factor-2 kinase protein kinase(NRS/TK) EGFR 1956 epidermal growth factor receptor (erythroblasticleukemia viral (v- protein kinase (RTK) erb-b) oncogene homolog, avian)EIF2AK1 27102 heme-regulated initiation factor 2-alpha kinase proteinkinase (NRS/TK) EIF2AK4 415116 serine/threonine-protein kinase pim-3protein kinase (RS/TK) EPHA1 2041 EPH receptor A1 protein kinase (RTK)EPHA2 1969 EPH receptor A2 protein kinase (RTK) EPHA3 2042 EPH receptorA3 protein kinase (RTK) EPHA4 2043 EPH receptor A4 protein kinase (RTK)EPHA6 285220 EPH receptor A6 protein kinase (RTK) EPHB1 2047 EPHreceptor B1 protein kinase (RTK) EPHB4 2050 EPH receptor B4 proteinkinase (RTK) EPHB6 2051 EPH receptor B6 protein kinase (RTK) ERBB2 2064v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, proteinkinase (RTK) neuro/glioblastoma derived oncogene homolog (avian) ERBB32065 v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 proteinkinase (RTK) (avian) ERBB4 2066 v-erb-a erythroblastic leukemia viraloncogene homolog 4 (avian) protein kinase (RTK) ERN1 2081 endoplasmicreticulum to nucleus signalling 1 protein kinase ETNK1 55500ethanolamine kinase 1 kinase related ETNK2 55224 ethanolamine kinase 2kinase related EXOSC10 5394 exosome component 10; EXOSC10 protein kinase(NRS/TK) FASTK 10922 FAST kinase protein kinase (NRS/TK) FASTKD1 79675FAST kinase domains 1; FASTKD1 protein kinase FASTKD2 22868 FAST kinasedomains 2; FASTKD2 protein kinase FASTKD3 79072 FAST kinase domains 3;FASTKD3 protein kinase FASTKD5 60493 FAST kinase domains 5; FASTKD5protein kinase FER 2241 fer (fps/fes related) tyrosine kinase(phosphoprotein NCP94) protein kinase (NRTK) FES 2242 feline sarcomaoncogene protein kinase (NRTK) FGFR1 2260 fibroblast growth factorreceptor 1 (fms-related tyrosine kinase 2, protein kinase (RTK) Pfeiffersyndrome) FGFR2 2263 fibroblast growth factor receptor 2 (craniofacialdysostosis 1, protein kinase (RTK) Crouzon, Pfeiffer and Jackson-Weisssyndrome) FGFR3 2261 fibroblast growth factor receptor 3(achondroplasia, thanatophoric protein kinase (RTK) dwarfism) FGFRL153834 fibroblast growth factor receptor-like 1; FGFRL1 protein kinase(RTK) FGR 2268 Gardner-Rasheed feline sarcoma viral (v-fgr) oncogenehomolog protein kinase (NRTK) FLJ10986 55277 hypothetical proteinFLJ10986; FLJ10986 carbohydrate kinase FLJ23356 84197 hypotheticalprotein FLJ23356 protein kinase FLJ25006 124923 hypothetical proteinFLJ25006 protein kinase (NRS/TK) FLJ40852 285962 hypothetical proteinFLJ40852 protein kinase FLT1 2321 fms-related tyrosine kinase 1(vascular endothelial growth protein kinase (RTK) factor/vascularpermeability factor receptor) FLT3 2322 fms-related tyrosine kinase 3protein kinase (RTK) FLT4 2324 fms-related tyrosine kinase 4 proteinkinase (RTK) FN3K 64122 fructosamine 3 kinase kinase related FN3KRP79672 fructosamine-3-kinase-related protein kinase related FRK 2444fyn-related kinase protein kinase (NRTK) FUK 197258 fucokinase kinaserelated FXN 2395 frataxin; FXN kinase related FYN 2534 FYN oncogenerelated to SRC, FGR, YES protein kinase (NRTK) GALK1 2584 galactokinase1 carbohydrate kinase GALK2 2585 galactokinase 2 carbohydrate kinase GCK2645 glucokinase (hexokinase 4, maturity onset diabetes of the youngcarbohydrate kinase 2) GK 2710 glycerol kinase carbohydrate kinase GK22712 glycerol kinase 2 carbohydrate kinase GK5 256356 hypotheticalprotein MGC40579; MGC40579 carbohydrate kinase GLYCTK 132158 glyceratekinase carbohydrate kinase GNE 10020 glucosamine(UDP-N-acetyl)-2-epimerase/N-acetylmannosamine carbohydrate kinasekinase GRK6 2870 G protein-coupled receptor kinase 6 protein kinase(NRS/TK) GRK7 131890 G protein-coupled receptor kinase 7 protein kinase(NRS/TK) GSG2 83903 haspin protein kinase (RS/TK) GSK3A 2931 glycogensynthase kinase 3 alpha protein kinase (NRS/TK) GTF2H1 2965 generaltranscription factor IIH, polypeptide 1, 62 kDa; GTF2H1 protein kinase(NRS/TK) GUK1 2987 guanylate kinase 1 nucleotide kinase HCK 3055hemopoietic cell kinase protein kinase (NRTK) HIPK1 204851 homeodomaininteracting protein kinase 1 protein kinase (NRS/TK) HIPK2 28996homeodomain interacting protein kinase 2 protein kinase (NRS/TK) HIPK310114 homeodomain interacting protein kinase 3 protein kinase (NRS/TK)HIPK4 147746 homeodomain interacting protein kinase 4 protein kinase(NRS/TK) HK1 3098 hexokinase 1 carbohydrate kinase HK2 3099 hexokinase 2carbohydrate kinase HK3 3101 hexokinase 3 (white cell) carbohydratekinase HKDC1 80201 hexokinase domain containing 1; HKDC1 carbohydratekinase HSPB8 26353 heat shock 22 kDa protein 8 protein kinase (NRS/TK)IGF1R 3480 insulin-like growth factor 1 receptor protein kinase (RTK)IHPK1 9807 inositol hexaphosphate kinase 1 kinase related IHPK2 51447inositol hexaphosphate kinase 2 Lipid Kinase IHPK3 117283 inositolhexaphosphate kinase 3 Lipid Kinase IKBKE 9641 inhibitor of kappa lightpolypeptide gene enhancer in B-cells, protein kinase kinase epsilon(NRS/TK) ILK 3611 integrin-linked kinase protein kinase (NRS/TK) INSRR3645 insulin receptor-related receptor protein kinase (RTK) IPMK 253430inositol polyphosphate multikinase Lipid Kinase IPPK 64768 inositol1,3,4,5,6-pentakisphosphate 2-kinase; IPPK Lipid Kinase IRAK2 3656interleukin-1 receptor-associated kinase 2 protein kinase (RS/TK) IRAK311213 interleukin-1 receptor-associated kinase 3 protein kinase (RS/TK)IRAK4 51135 interleukin-1 receptor-associated kinase 4 protein kinase(RS/TK) ITGB1BP3 27231 integrin beta 1 binding protein 3; ITGB1BP3kinase related ITK 3702 IL2-inducible T-cell kinase protein kinase(NRTK) ITPKB 3707 inositol 1,4,5-trisphosphate 3-kinase B kinase relatedJAK1 3716 Janus kinase 1 (a protein tyrosine kinase) protein kinase(NRTK) JAK2 3717 Janus kinase 2 (a protein tyrosine kinase) proteinkinase (NRTK) JAK3 3718 Janus kinase 3 (a protein tyrosine kinase,leukocyte) protein kinase (NRTK) KDR 3791 kinase insert domain receptor(a type III receptor tyrosine kinase) protein kinase (RTK) KHK 3795ketohexokinase (fructokinase) carbohydrate kinase KIAA0999 23387KIAA0999 protein protein kinase (RS/TK) KIAA2002 79834 KIAA2002 proteinprotein kinase (RS/TK) KSR 8844 kinase suppressor of ras protein kinase(NRS/TK) KSR2 283455 kinase suppressor of Ras-2 protein kinase (NRS/TK)LATS1 9113 LATS, large tumor suppressor, homolog 1 (Drosophila) proteinkinase (NRS/TK) LATS2 26524 LATS, large tumor suppressor, homolog 2(Drosophila) protein kinase (NRS/TK) LCK 3932 lymphocyte-specificprotein tyrosine kinase protein kinase (NRTK) LIMK1 3984 LIM domainkinase 1 protein kinase (NRS/TK) LIMK2 3985 LIM domain kinase 2 proteinkinase (NRS/TK) LMTK2 22853 lemur tyrosine kinase 2 protein kinase (RTK)LOC220686 220686 hypothetical protein LOC220686; LOC220686 kinaserelated LOC340156 340156 hypothetical protein LOC340156 protein kinase(NRS/TK) LOC340371 340371 hypothetical protein LOC340371 protein kinase(NRS/TK) LOC375133 375133 similar to phosphatidylinositol 4-kinase alphaLipid Kinase LOC388957 388957 similar to BMP2 inducible kinase proteinkinase (NRS/TK) LOC389599 389599 similar to amyotrophic lateralsclerosis 2 (juvenile) chr. region, protein kinase candidate 2;ILP-interacting protein ILPIPA LOC390877 390877 similar to adenylatekinase (EC 2.7.4.3), cytosolic - common carp nucleotide kinase LOC442075442075 similar to serine/threonine kinase, establishes embryonicpolarity protein kinase (NRS/TK) LOC54103 54103 hypothetical proteinLOC54103; LOC54103 protein kinase (RTK) LOC646505 646505 similar to Dualspecificity protein kinase CLK3 (CDC-like kinase protein kinase 3);unassigned (NRS/TK) LOC647279 647279 similar to MAP/microtubuleaffinity-regulating kinase protein kinase 3; unassigned (NRS/TK)LOC648152 648152 similar to ataxia telangiectasia and Rad3 relatedprotein kinase protein; unassigned (NRS/TK) LOC649288 649288 similar toAdenylate kinase isoenzyme 4, mitochondrial nucleotide kinase (Adenylatekinase 3-like 1) LOC650122 650122 similar to choline kinase alphaisoform a; unassigned kinase related LOC652722 652722 similar to PTK2protein tyrosine kinase 2 isoform a; unassigned protein kinase (NRTK)LOC652799 652799 similar to Mast/stem cell growth factor receptorprecursor (SCFR) protein kinase (RTK) (c-kit) (CD117 antigen);unassigned LOC653052 653052 similar to Homeodomain-interacting proteinkinase 2 protein kinase (hHIPk2); unassigned (NRS/TK) LOC653155 653155similar to PRP4 pre-mRNA processing factor 4 homolog protein kinase B;unassigned (NRS/TK) LOC727761 727761 similar to deoxythymidylate kinase(thymidylate nucleotide kinase kinase); unassigned LOC730000 730000similar to testis-specific serine kinase 6; unassigned protein kinase(NRS/TK) LOC732306 732306 similar to vaccinia related kinase 2;unassigned protein kinase (NRS/TK) LOC91461 91461 hypothetical proteinBC007901 protein kinase (NRTK) LOC91807 91807 myosin light chain kinase(MLCK) protein kinase (NRS/TK) LRGUK 136332 leucine-rich repeats andguanylate kinase domain nucleotide kinase containing; LRGUK LRPPRC 10128leucine-rich PPR-motif containing; LRPPRC protein kinase (RS/TK) LRRK2120892 leucine-rich repeat kinase 2 protein kinase (NRS/TK) LYK5 92335protein kinase LYK5 protein kinase LYN 4067 v-yes-1 Yamaguchi sarcomaviral related oncogene homolog protein kinase (NRTK) MAGI1 9223 membraneassociated guanylate kinase, WW and PDZ domain nucleotide kinasecontaining 1; MAGI1 MAK 4117 male germ cell-associated kinase proteinkinase (NRS/TK) MAP2K1 5604 mitogen-activated protein kinase kinase 1protein kinase MAP2K1IP1 8649 mitogen-activated protein kinase kinase 1interacting protein 1 protein kinase MAP2K2 5605 mitogen-activatedprotein kinase kinase 2 protein kinase MAP2K5 5607 mitogen-activatedprotein kinase kinase 5 protein kinase MAP2K6 5608 mitogen-activatedprotein kinase kinase 6 protein kinase MAP2K7 5609 mitogen-activatedprotein kinase kinase 7 protein kinase MAP3K11 4296 mitogen-activatedprotein kinase kinase kinase 11 protein kinase (NRS/TK) MAP3K12 7786mitogen-activated protein kinase kinase kinase 12 protein kinase(NRS/TK) MAP3K14 9020 mitogen-activated protein kinase kinase kinase 14protein kinase MAP3K15 389840 FLJ16518 protein protein kinase MAP3K210746 mitogen-activated protein kinase kinase kinase 2 protein kinaseMAP3K5 4217 mitogen-activated protein kinase kinase kinase 5 proteinkinase MAP3K6 9064 mitogen-activated protein kinase kinase kinase 6protein kinase MAP3K7 6885 mitogen-activated protein kinase kinasekinase 7 protein kinase (NRS/TK) MAP3K8 1326 mitogen-activated proteinkinase kinase kinase 8 protein kinase MAP4K1 11184 mitogen-activatedprotein kinase kinase kinase kinase 1 protein kinase MAP4K2 5871mitogen-activated protein kinase kinase kinase kinase 2 protein kinaseMAP4K3 8491 mitogen-activated protein kinase kinase kinase kinase 3protein kinase MAP4K4 9448 mitogen-activated protein kinase kinasekinase kinase 4 protein kinase MAP4K5 11183 mitogen-activated proteinkinase kinase kinase kinase 5 protein kinase MAPK1 5594mitogen-activated protein kinase 1 protein kinase (NRS/TK) MAPK10 5602mitogen-activated protein kinase 10 protein kinase (NRS/TK) MAPK12 6300mitogen-activated protein kinase 12 protein kinase (NRS/TK) MAPK13 5603mitogen-activated protein kinase 13 protein kinase (NRS/TK) MAPK14 1432mitogen-activated protein kinase 14 protein kinase (NRS/TK) MAPK15225689 extracellular signal-regulated kinase 8 protein kinase (NRS/TK)MAPK3 5595 mitogen-activated protein kinase 3 protein kinase (NRS/TK)MAPK4 5596 mitogen-activated protein kinase 4 protein kinase (NRS/TK)MAPK6 5597 mitogen-activated protein kinase 6 protein kinase (NRS/TK)MAPK8 5599 mitogen-activated protein kinase 8 protein kinase (NRS/TK)MAPK9 5601 mitogen-activated protein kinase 9 protein kinase (NRS/TK)MAPKAPK2 9261 mitogen-activated protein kinase-activated protein kinase2 protein kinase (NRS/TK) MAPKAPK3 7867 mitogen-activated proteinkinase-activated protein kinase 3 protein kinase (NRS/TK) MAPKAPK5 8550mitogen-activated protein kinase-activated protein kinase 5 proteinkinase (NRS/TK) MARK2 2011 MAP/microtubule affinity-regulating kinase 2protein kinase (NRS/TK) MARK3 4140 MAP/microtubule affinity-regulatingkinase 3 protein kinase (NRS/TK) MAST1 22983 microtubule associatedserine/threonine kinase 1 protein kinase (NRS/TK) MAST2 23139microtubule associated serine/threonine kinase 2 protein kinase (NRS/TK)MASTL 84930 microtubule associated serine/threonine kinase-like proteinkinase (NRS/TK) MATK 4145 megakaryocyte-associated tyrosine kinaseprotein kinase (NRTK) MERTK 10461 c-mer proto-oncogene tyrosine kinaseprotein kinase (RTK) MET 4233 met proto-oncogene (hepatocyte growthfactor receptor) protein kinase (RTK) MGC16169 93627 hypotheticalprotein MGC16169 protein kinase MGC42105 167359 hypothetical proteinMGC42105 protein kinase (NRS/TK) MINK1 50488 misshapen/NIK-relatedkinase protein kinase MKNK1 8569 MAP kinase interacting serine/threoninekinase 1 protein kinase (NRS/TK) MKNK2 2872 MAP kinase interactingserine/threonine kinase 2 protein kinase (NRS/TK) MORN2 378464 MORNrepeat containing 2; MORN2 kinase related MOS 4342 v-mos Moloney murinesarcoma viral oncogene homolog protein kinase (NRS/TK) MPP1 4354membrane protein, palmitoylated 1, 55 kDa; MPP1 protein kinase MPP2 4355membrane protein, palmitoylated 2 (MAGUK p55 subfamily nucleotide kinasemember 2); MPP2 MPP3 4356 membrane protein, palmitoylated 3 (MAGUK p55subfamily nucleotide kinase member 3); MPP3 MPP4 58538 membrane protein,palmitoylated 4 (MAGUK p55 subfamily nucleotide kinase member 4); MPP4MPP5 64398 membrane protein, palmitoylated 5 (MAGUK p55 subfamilynucleotide kinase member 5); MPP5 MPP6 51678 membrane protein,palmitoylated 6 (MAGUK p55 subfamily nucleotide kinase member 6); MPP6MPP7 143098 membrane protein, palmitoylated 7 (MAGUK p55 subfamilynucleotide kinase member 7); MPP7 MST1R 4486 macrophage stimulating 1receptor (c-met-related tyrosine protein kinase (RTK) kinase) MUSK 4593muscle, skeletal, receptor tyrosine kinase protein kinase (RTK) MVK 4598mevalonate kinase (mevalonic aciduria) kinase related MYLK2 85366 myosinlight chain kinase 2, skeletal muscle protein kinase (NRS/TK) MYO3B140469 myosin IIIB protein kinase (NRS/TK) NADK 65220 NAD kinase kinaserelated NAGK 55577 N-acetylglucosamine kinase kinase related NEK10152110 NIMA (never in mitosis gene a)-related kinase 10 protein kinaseNEK11 79858 NIMA (never in mitosis gene a)-related kinase 11 proteinkinase NEK2 4751 NIMA (never in mitosis gene a)-related kinase 2 proteinkinase NEK3 4752 NIMA (never in mitosis gene a)-related kinase 3 proteinkinase NEK4 6787 NIMA (never in mitosis gene a)-related kinase 4 proteinkinase NEK5 341676 NIMA (never in mitosis gene a)-related kinase 5protein kinase NEK6 10783 NIMA (never in mitosis gene a)-related kinase6 protein kinase NEK7 140609 NIMA (never in mitosis gene a)-relatedkinase 7 protein kinase NEK8 284086 NIMA (never in mitosis genea)-related kinase 8 protein kinase NEK9 91754 NIMA (never in mitosisgene a)-related kinase 9 protein kinase NJMU-R1 64149 protein kinaseNjmu-R1 protein kinase NLK 51701 nemo like kinase protein kinase(NRS/TK) NME1 4830 nucleoside-diphosphate kinase 1 nucleotide kinaseNME1-NME2 654364 NME1-NME2 protein; NME1-NME2 nucleotide kinase NME24831 nucleoside-diphosphate kinase 2 nucleotide kinase NME3 4832nucleoside-diphosphate kinase 3 nucleotide kinase NME4 4833nucleoside-diphosphate kinase 4 nucleotide kinase NME5 8382non-metastatic cells 5, protein expressed in (nucleoside- nucleotidekinase diphosphate kinase) NME6 10201 non-metastatic cells 6, proteinexpressed in (nucleoside- nucleotide kinase diphosphate kinase) NME729922 non-metastatic cells 7, protein expressed in (nucleoside-nucleotide kinase diphosphate kinase) NPR2 4882 natriuretic peptidereceptor B/guanylate cyclase B (atrionatriuretic protein kinase peptidereceptor B) NRBP 29959 nuclear receptor binding protein protein kinase(NRS/TK) NTRK1 4914 neurotrophic tyrosine kinase, receptor, type 1protein kinase (RTK) NTRK2 4915 neurotrophic tyrosine kinase, receptor,type 2 protein kinase (RTK) NTRK3 4916 neurotrophic tyrosine kinase,receptor, type 3 protein kinase (RTK) NUAK2 81788 likely ortholog of ratSNF1/AMP-activated protein kinase protein kinase (NRS/TK) NUP62 23636nucleoporin 62 kDa; NUP62 protein kinase (NRS/TK) NYD- 89882 proteinkinase NYD-SP25 protein kinase SP25 OXSR1 9943 oxidative-stressresponsive 1 protein kinase PAK1 5058 p21/Cdc42/Rac1-activated kinase 1(STE20 homolog, yeast) protein kinase PAK2 5062 p21 (CDKN1A)-activatedkinase 2 protein kinase PAK3 5063 p21 (CDKN1A)-activated kinase 3protein kinase PAK4 10298 p21(CDKN1A)-activated kinase 4 protein kinasePAK6 56924 p21(CDKN1A)-activated kinase 6 protein kinase PAK7 57144p21(CDKN1A)-activated kinase 7 protein kinase PANK2 80025 pantothenatekinase 2 (Hallervorden-Spatz syndrome) kinase related PANK3 79646pantothenate kinase 3 kinase related PANK4 55229 pantothenate kinase 4kinase related PAPSS1 9061 3′-phosphoadenosine 5′-phosphosulfatesynthase 1; PAPSS1 kinase related PAPSS2 9060 3′-phosphoadenosine5′-phosphosulfate synthase 2; PAPSS2 kinase related PBK 55872 T-LAKcell-originated protein kinase protein kinase PCK2 5106phosphoenolpyruvate carboxykinase 2 (mitochondrial) kinase related PCTK15127 PCTAIRE protein kinase 1 protein kinase (NRS/TK) PCTK2 5128 PCTAIREprotein kinase 2 protein kinase (NRS/TK) PCTK3 5129 PCTAIRE proteinkinase 3 protein kinase (NRS/TK) PDGFRA 5156 platelet-derived growthfactor receptor, alpha polypeptide protein kinase (RTK) PDGFRB 5159platelet-derived growth factor receptor, beta polypeptide protein kinase(RTK) PDGFRL 5157 platelet-derived growth factor receptor-like; PDGFRLprotein kinase (RTK) PDIK1L 149420 PDLIM1 interacting kinase 1 likeprotein kinase (NRS/TK) PDK1 5163 pyruvate dehydrogenase kinase,isoenzyme 1 protein kinase PDK2 5164 pyruvate dehydrogenase kinase,isoenzyme 2 protein kinase PDK3 5165 pyruvate dehydrogenase kinase,isoenzyme 3 protein kinase PDK4 5166 pyruvate dehydrogenase kinase,isoenzyme 4 protein kinase PDPK1 5170 3-phosphoinositide dependentprotein kinase-1 protein kinase PDXK 8566 pyridoxal (pyridoxine, vitaminB6) kinase kinase related PFKFB1 52076-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 carbohydratekinase PFKFB2 5208 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2carbohydrate kinase PFKFB3 52096-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 carbohydratekinase PFKL 5211 phosphofructokinase, liver carbohydrate kinase PFKM5213 phosphofructokinase, muscle carbohydrate kinase PFKP 5214phosphofructokinase, platelet carbohydrate kinase PFTK1 5218 PFTAIREprotein kinase 1 protein kinase (NRS/TK) PGK1 5230 phosphoglyceratekinase 1 carbohydrate kinase PGK2 5232 phosphoglycerate kinase 2carbohydrate kinase PHKA1 5255 phosphorylase kinase, alpha 1 (muscle)protein kinase (NRS/TK) PHKA2 5256 phosphorylase kinase, alpha 2 (liver)protein kinase (NRS/TK) PHKB 5257 phosphorylase kinase, beta proteinkinase (NRS/TK) PHKG1 5260 phosphorylase kinase, gamma 1 (muscle)protein kinase (NRS/TK) PHKG2 5261 phosphorylase kinase, gamma 2(testis) protein kinase (NRS/TK) PI4K2B 55300 phosphatidylinositol4-kinase type-II beta Lipid Kinase PI4KII 55361 phosphatidylinositol4-kinase type II Lipid Kinase PIK3C2G 5288 phosphoinositide-3-kinase,class 2, gamma polypeptide Lipid Kinase PIK3C3 5289phosphoinositide-3-kinase, class 3 Lipid Kinase PIK3CA 5290phosphoinositide-3-kinase, catalytic, alpha polypeptide Lipid KinasePIK3CBP 5291 phosphoinositide-3-kinase, catalytic, beta polypeptideLipid Kinase PIK3CG 5294 phosphoinositide-3-kinase, catalytic, gammapolypeptide Lipid Kinase PIK3R1 5295 phosphoinositide-3-kinase,regulatory subunit 1 (p85 alpha) Lipid Kinase PIK3R3 8503phosphoinositide-3-kinase, regulatory subunit 3 (p55, gamma) LipidKinase PIK3R4 30849 phosphoinositide-3-kinase, regulatory subunit 4,p150 Lipid Kinase PIK3R5 23533 phosphoinositide-3-kinase, regulatorysubunit 5, p101 Lipid Kinase PIK4CA 5297 phosphatidylinositol 4-kinase,catalytic, alpha polypeptide Lipid Kinase PIK4CB 5298phosphatidylinositol 4-kinase, catalytic, beta polypeptide Lipid KinasePIM1 5292 pim-1 oncogene protein kinase (RS/TK) PIM2 11040 pim-2oncogene protein kinase (RS/TK) PINK1 65018 PTEN induced putative kinase1 protein kinase (RS/TK) PIP5K1A 8394 phosphatidylinositol-4-phosphate5-kinase, type I, alpha Lipid Kinase PIP5K1B 8395phosphatidylinositol-4-phosphate 5-kinase, type I, beta Lipid KinasePIP5K2A 5305 phosphatidylinositol-4-phosphate 5-kinase, type II, alphaLipid Kinase PIP5K2C 79837 phosphatidylinositol-4-phosphate 5-kinase,type II, gamma kinase related PIP5K3 200576phosphatidylinositol-3-phosphate/phosphatidylinositol 5-kinase, LipidKinase type III PIP5KL1 138429 phosphatidylinositol-4-phosphate5-kinase-like 1 Lipid Kinase PKLR 5313 pyruvate kinase, liver and RBCcarbohydrate kinase PKM2 5315 pyruvate kinase, muscle carbohydratekinase PKMYT1 9088 membrane-associated tyrosine- and threonine-specificcdc2- protein kinase inhibitory kinase (NRS/TK) PLAU 5328 plasminogenactivator, urokinase kinase related PLK1 5347 polo-like kinase 1(Drosophila) protein kinase (NRS/TK) PLK2 10769 polo-like kinase 2(Drosophila) protein kinase (NRS/TK) PLK4 10733 polo-like kinase 4(Drosophila) protein kinase (NRS/TK) PLXNA3 55558 plexin A3; PLXNA3protein kinase (RTK) PLXNA4B 91584 plexin A4, B; PLXNA4B protein kinase(RTK) PLXNB2 23654 plexin B2; PLXNB2 protein kinase (RTK) PMVK 10654phosphomevalonate kinase kinase related PNCK 139728 pregnancyupregulated non-ubiquitously expressed CaM kinase protein kinase(NRS/TK) PNKP 11284 polynucleotide kinase 3′-phosphatase nucleotidekinase PRKAA1 5562 protein kinase, AMP-activated, alpha 1 catalyticsubunit protein kinase (NRS/TK) PRKAA2 5563 protein kinase,AMP-activated, alpha 2 catalytic subunit protein kinase (NRS/TK) PRKAB15564 protein kinase, AMP-activated, beta 1 non-catalytic subunit proteinkinase (NRS/TK) PRKAB2 5565 protein kinase, AMP-activated, beta 2non-catalytic subunit protein kinase (NRS/TK) PRKACA 5566 proteinkinase, cAMP-dependent, catalytic, alpha protein kinase (NRS/TK) PRKACB5567 protein kinase, cAMP-dependent, catalytic, beta protein kinase(NRS/TK) PRKACG 5568 protein kinase, cAMP-dependent, catalytic, gammaprotein kinase (NRS/TK) PRKAG1 5571 protein kinase, AMP-activated, gamma1 non-catalytic subunit protein kinase (NRS/TK) PRKAG2 51422 proteinkinase, AMP-activated, gamma 2 non-catalytic subunit protein kinase(NRS/TK) PRKAG3 53632 protein kinase, AMP-activated, gamma 3non-catalytic subunit protein kinase (NRS/TK) PRKAR1A 5573 proteinkinase, cAMP-dependent, regulatory, type I, alpha (tissue protein kinasespecific extinguisher 1) (NRS/TK) PRKAR1B 5575 protein kinase,cAMP-dependent, regulatory, type I, beta protein kinase (NRS/TK) PRKAR2A5576 protein kinase, cAMP-dependent, regulatory, type II, alpha proteinkinase (NRS/TK) PRKAR2B 5577 protein kinase, cAMP-dependent, regulatory,type II, beta protein kinase (NRS/TK) PRKCA 5578 protein kinase C, alphaprotein kinase (NRS/TK) PRKCB1 5579 protein kinase C, beta 1 proteinkinase (NRS/TK) PRKCE 5581 protein kinase C, epsilon protein kinase(NRS/TK) PRKCG 5582 protein kinase C, gamma protein kinase (NRS/TK)PRKCH 5583 protein kinase C, eta protein kinase (NRS/TK) PRKCI 5584protein kinase C, iota protein kinase (NRS/TK) PRKCQ 5588 protein kinaseC, theta protein kinase (NRS/TK) PRKCZ 5590 protein kinase C, zetaprotein kinase (NRS/TK) PRKD1 5587 protein kinase D1 protein kinase(NRS/TK) PRKD2 25865 protein kinase D2 protein kinase (NRS/TK) PRKD323683 protein kinase D3 protein kinase (NRS/TK) PRKG1 5592 proteinkinase, cGMP-dependent, type I protein kinase (NRS/TK) PRKG2 5593protein kinase, cGMP-dependent, type II protein kinase (NRS/TK) PRKR5610 protein kinase, interferon-inducible double stranded RNA proteinkinase dependent (NRS/TK) PRKX 5613 protein kinase, X-linked proteinkinase (NRS/TK) PRKY 5616 protein kinase, Y-linked protein kinase(NRS/TK) PRPF4B 8899 PRP4 pre-mRNA processing factor 4 homolog B (yeast)protein kinase (NRS/TK) PRPS1 5631 phosphoribosyl pyrophosphatesynthetase 1; PRPS1 kinase related PRPS1L1 221823 phosphoribosylpyrophosphate synthetase 1-like 1; PRPS1L1 kinase related PRPS2 5634phosphoribosyl pyrophosphate synthetase 2; PRPS2 kinase related PSKH15681 protein serine kinase H1 protein kinase (NRS/TK) PTK2 5747 PTK2protein tyrosine kinase 2 protein kinase (NRTK) PTK2B 2185 PTK2B proteintyrosine kinase 2 beta protein kinase (NRTK) PTK6 5753 PTK6 proteintyrosine kinase 6 protein kinase (NRTK) PTK7 5754 PTK7 protein tyrosinekinase 7 protein kinase (RTK) PTK9 5756 PTK9 protein tyrosine kinase 9protein kinase (NRTK) PXK 54899 PX domain containing serine/threoninekinase kinase related RAF1 5894 v-raf-1 murine leukemia viral oncogenehomolog 1 protein kinase (NRS/TK) RAGE 5891 renal tumor antigen proteinkinase (NRS/TK) RBKS 64080 ribokinase nucleotide kinase RET 5979 retproto-oncogene (multiple endocrine neoplasia and medullary proteinkinase (RTK) thyroid carcinoma 1, Hirschsprung disease) RFK 55312riboflavin kinase kinase related RIOK1 83732 RIO kinase 1 (yeast)protein kinase (NRS/TK) RIOK2 55781 RIO kinase 2 (yeast) protein kinase(NRS/TK) RIOK3 8780 RIO kinase 3 (yeast) protein kinase (NRS/TK) RIPK18737 receptor (TNFRSF)-interacting serine-threonine kinase 1 proteinkinase (NRS/TK) RIPK2 8767 receptor-interacting serine-threonine kinase2 protein kinase (NRS/TK) RIPK5 25778 receptor interacting proteinkinase 5 protein kinase (NRS/TK) RKHD3 84206 ring finger and KH domaincontaining 3; RKHD3 protein kinase ROR2 4920 receptor tyrosinekinase-like orphan receptor 2 protein kinase (RTK) RP2 6102 retinitispigmentosa 2 (X-linked recessive); RP2 nucleotide kinase RP6- 51765 Mst3and SOK1-related kinase protein kinase 213H19.1 RPS6KA1 6195 ribosomalprotein S6 kinase, 90 kDa, polypeptide 1 protein kinase (NRS/TK) RPS6KA26196 ribosomal protein S6 kinase, 90 kDa, polypeptide 2 protein kinase(NRS/TK) RPS6KA3 6197 ribosomal protein S6 kinase, 90 kDa, polypeptide 3protein kinase (NRS/TK) RPS6KA4 8986 ribosomal protein S6 kinase, 90kDa, polypeptide 4 protein kinase (NRS/TK) RPS6KA5 9252 ribosomalprotein S6 kinase, 90 kDa, polypeptide 5 protein kinase (NRS/TK) RPS6KA627330 ribosomal protein S6 kinase, 90 kDa, polypeptide 6 protein kinase(NRS/TK) RPS6KB1 6198 ribosomal protein S6 kinase, 70 kDa, polypeptide 1protein kinase (NRS/TK) RPS6KB2 6199 ribosomal protein S6 kinase, 70kDa, polypeptide 2 protein kinase (NRS/TK) RPS6KC1 26750 ribosomalprotein S6 kinase, 52 kDa, polypeptide 1 protein kinase (NRS/TK) RPS6KL183694 ribosomal protein S6 kinase-like 1 protein kinase (NRS/TK) SCYL157410 SCY1-like 1 (S. cerevisiae) protein kinase (NRTK) SCYL2 55681hypothetical protein FLJ10074 protein kinase (NRTK) SCYL3 57147ezrin-binding partner PACE-1 protein kinase (NRTK) SEPHS2 22928selenophosphate synthetase 2; SEPHS2 kinase related SGK 6446serum/glucocorticoid regulated kinase protein kinase (NRS/TK) SGK2 10110serum/glucocorticoid regulated kinase 2 protein kinase (NRS/TK) SGK323678 serum/glucocorticoid regulated kinase-like protein kinase (NRS/TK)SH3BP4 23677 SH3-domain binding protein 4; SH3BP4 protein kinase (NRTK)SH3BP5 9467 SH3-domain binding protein 5 (BTK-associated); SH3BP5 kinaserelated SH3BP5L 80851 SH3-binding domain protein 5-like; SH3BP5L kinaserelated SLAMF6 114836 SLAM family member 6; SLAMF6 protein kinase (RTK)SNF1LK 150094 SNF1-like kinase protein kinase (NRS/TK) SNRK 54861 SNF-1related kinase protein kinase (NRS/TK) SNX16 64089 sorting nexin 16;SNX16 kinase related SPHK1 8877 sphingosine kinase 1 carbohydrate kinaseSPHK2 56848 sphingosine kinase 2 carbohydrate kinase SRC 6714 v-srcsarcoma (Schmidt-Ruppin A-2) viral oncogene homolog protein kinase(NRTK) (avian) SRPK1 6732 SFRS protein kinase 1 protein kinase (NRS/TK)SRPK2 6733 SFRS protein kinase 2 protein kinase (NRS/TK) SRPK3 26576serine/threonine kinase 23 protein kinase (NRS/TK) STK11 6794serine/threonine kinase 11 (Peutz-Jeghers syndrome) protein kinase(NRS/TK) STK16 8576 serine/threonine kinase 16 protein kinase (NRS/TK)STK17B 9262 serine/threonine kinase 17b (apoptosis-inducing) proteinkinase (NRS/TK) STK19 8859 serine/threonine kinase 19 protein kinase(NRS/TK) STK24 8428 serine/threonine kinase 24 (STE20 homolog, yeast)protein kinase STK25 10494 serine/threonine kinase 25 (STE20 homolog,yeast) protein kinase STK3 6788 serine/threonine kinase 3 (STE20homolog, yeast) protein kinase STK32A 202374 serine/threonine kinase 32Aprotein kinase (NRS/TK) STK32B 55351 serine/threonine kinase 32B proteinkinase (NRS/TK) STK32C 282974 serine/threonine kinase 32C protein kinase(NRS/TK) STK33 65975 serine/threonine kinase 33 protein kinase (NRS/TK)STK36 27148 serine/threonine kinase 36 (fused homolog, Drosophila)protein kinase (RS/TK) STK38 11329 serine/threonine kinase 38 proteinkinase (NRS/TK) STK38L 23012 serine/threonine kinase 38 like proteinkinase (NRS/TK) STK40 83931 Ser/Thr-like kinase protein kinase STYK155359 protein kinase STYK1 protein kinase (RTK) SYK 6850 spleen tyrosinekinase protein kinase (NRTK) TAOK3 51347 TAO kinase 3 protein kinaseTBK1 29110 TANK-binding kinase 1 protein kinase (NRS/TK) TEC 7006 tecprotein tyrosine kinase protein kinase (NRTK) TESK1 7016 testis-specifickinase 1 protein kinase (NRS/TK) TESK2 10420 testis-specific kinase 2protein kinase (NRS/TK) TGFBR1 7046 transforming growth factor, betareceptor I (activin A receptor protein kinase (RS/TK) type II-likekinase, 53 kDa) TGFBR2 7048 transforming growth factor, beta receptor II(70/80 kDa) protein kinase (RS/TK) TGFBR3 7049 transforming growthfactor, beta receptor III (betaglycan, protein kinase (RS/TK) 300 kDa);TGFBR3 TIE1 7075 tyrosine kinase with immunoglobulin-like and EGF-likedomains 1 protein kinase (RTK) TK1 7083 thymidine kinase 1, solublenucleotide kinase TLK1 9874 tousled-like kinase 1 protein kinase(NRS/TK) TLK2 11011 tousled-like kinase 2 protein kinase (NRS/TK) TNK18711 tyrosine kinase, non-receptor, 1 protein kinase (NRTK) TNNI3K 51086TNNI3 interacting kinase protein kinase TP53RK 112858 TP53 regulatingkinase protein kinase (NRS/TK) TPK1 27010 thiamin pyrophosphokinase 1kinase related TPR 7175 translocated promoter region (to activated METoncogene); TPR protein kinase (RTK) TRIB1 10221 tribbles homolog 1(Drosophila) protein kinase TRIB2 28951 tribbles homolog 2 (Drosophila)protein kinase TRIB3 57761 tribbles homolog 3 (Drosophila) proteinkinase TRIM27 5987 tripartite motif-containing 27; TRIM27 protein kinase(RTK) TRPM7 54822 transient receptor potential cation channel, subfamilyM, member 7 protein kinase (NRS/TK) TSSK1 83942 serine/threonine kinase22D (spermiogenesis associated) protein kinase (NRS/TK) TSSK2 23617serine/threonine kinase 22B (spermiogenesis associated) protein kinase(NRS/TK) TSSK3 81629 serine/threonine kinase 22C (spermiogenesisassociated) protein kinase (NRS/TK) TSSK6 83983 serine/threonine proteinkinase SSTK protein kinase (NRS/TK) TTK 7272 TTK protein kinase proteinkinase TWF2 11344 PTK9L protein tyrosine kinase 9-like (A6-relatedprotein) protein kinase (NRTK) TXK 7294 TXK tyrosine kinase proteinkinase (NRTK) TXNDC3 51314 thioredoxin domain containing 3(spermatozoa); TXNDC3 nucleotide kinase TYK2 7297 tyrosine kinase 2protein kinase (NRTK) TYRO3 7301 TYRO3 protein tyrosine kinase proteinkinase (RTK) UCK2 7371 uridine-cytidine kinase 2 nucleotide kinase UHMK1127933 U2AF homology motif (UHM) kinase 1 protein kinase (NRS/TK) ULK29706 unc-51-like kinase 2 (C. elegans) protein kinase (NRS/TK) ULK325989 DKFZP434C131 protein protein kinase (NRS/TK) ULK4 54986hypothetical protein FLJ20574 protein kinase VRK1 7443 vaccinia relatedkinase 1 protein kinase (NRS/TK) VRK2 7444 vaccinia related kinase 2protein kinase (NRS/TK) VRK3 51231 vaccinia related kinase 3 proteinkinase (NRS/TK) WNK1 65125 protein kinase, lysine deficient 1 proteinkinase (NRS/TK) WNK4 65266 protein kinase, lysine deficient 4 proteinkinase (NRS/TK) XRCC6BP1 91419 XRCC6 binding protein 1; XRCC6BP1 proteinkinase XYLB 9942 xylulokinase homolog (H. influenzae) carbohydratekinase YES1 7525 v-yes-1 Yamaguchi sarcoma viral oncogene homolog 1protein kinase (NRTK) YSK4 80122 hypothetical protein FLJ23074 proteinkinase ZAK 51776 sterile alpha motif and leucine zipper containingkinase AZK protein kinase (NRS/TK) ZAP70 7535 zeta-chain (TCR)associated protein kinase 70 kDa protein kinase (NRTK) Abbreviations:RS/TK (Receptor Serine/Threonine Kinase); RTK (Receptor TyrosineKinase); NRS/TK(Non-Receptor Serine/Threonine Kinase); NRTK(Non-Receptor Tyrosine Kinase)

TABLE 4 Ranking of average differential proliferation (1 μMPLX4720/control) for 597 kinase-related ORFs, relative to MEK DD Z- RANKGENE AVERAGE STDEV. SCORE NOTES — ALL ORFS 44.26% 7.19% — 1 MEK DD100.00% 6.54% 7.75 (+) Control 2 RAF1 86.80% 18.00% 5.92 3 PRKCE 74.76%8.78% 4.24 4 MAP3K8 67.51% 10.03% 3.23 5 PRKCH 67.37% 17.56% 3.21 6 FGR65.03% 20.76% 2.89 7 CRKL 64.74% 19.66% 2.85 8 PAK3 60.33% 13.59% 2.23 9AXL 59.75% 10.23% 2.15 10 LCK 59.47% 15.66% 2.11 Lethal 11 ERBB2 59.32%9.39% 2.09 2 SD = 58.64% 12 PRKCQ 55.40% 11.97% 1.55 13 NME3 55.10%15.46% 1.51 14 MOS 55.04% 11.84% 1.50 15 KHK 55.03% 14.75% 1.50 16 TIE154.65% 15.26% 1.44 Prolifer- ative 17 PRKAG2 54.23% 7.15% 1.39 18LOC91461 53.75% 12.45% 1.32 19 TYRO3 53.64% 10.57% 1.30 20 CDK3 53.55%13.92% 1.29 21 PIM2 53.36% 10.78% 1.27 22 CIB4 53.31% 12.21% 1.26 23PRPS2 53.24% 14.53% 1.25 24 PRKCB1 53.12% 7.74% 1.23 25 ACVR1B 53.08%9.53% 1.23 26 ETNK2 52.85% 12.63% 1.19 27 STK36 52.85% 15.28% 1.19 28DDR1 52.78% 15.54% 1.19 29 PRKCA 52.47% 13.38% 1.14 30 AURKB 52.22%11.80% 1.11 31 CAMK4 52.14% 9.09% 1.10 32 DAPK1 51.94% 11.43% 1.07 33AURKC 51.89% 11.23% 1.06 Prolifer- ative 34 IGF1R 51.35% 10.70% 0.99 35FN3K 51.27% 12.65% 0.97 36 LOC646505 50.97% 16.89% 0.93 37 PIK3CB 50.90%12.87% 0.92 38 YES1 50.68% 9.51% 0.89 39 LOC340156 50.59% 14.63% 0.88 40MAP4K5 50.57% 35.45% 0.88 41 ABL1 50.55% 19.09% 0.87 42 MAP2K1IP1 50.47%11.54% 0.86 43 PRKAR1B 50.34% 8.31% 0.84 44 RPS6KA1 50.12% 12.28% 0.8145 TSSK6 50.10% 11.70% 0.81 46 NEK9 50.04% 14.37% 0.80 47 DLG1 49.98%12.07% 0.80 48 PTK6 49.93% 10.93% 0.79 49 SCYL2 49.85% 12.51% 0.78 50STK11 49.82% 12.10% 0.77 51 C9orf98 49.82% 10.38% 0.77 52 PCK2 49.77%12.93% 0.77 53 NTRK2 49.77% 10.02% 0.77 54 TRIM27 49.69% 10.38% 0.75 55FN3KRP 49.69% 11.83% 0.75 56 CAMK2D 49.61% 11.93% 0.74 57 ALPK2 49.56%10.41% 0.74 58 LOC652722 49.52% 13.10% 0.73 59 LATS2 49.49% 12.45% 0.7360 STK3 49.48% 10.15% 0.73 61 CDC42BPG 49.44% 10.19% 0.72 62 HKDC149.44% 11.53% 0.72 63 CDK7 49.42% 14.45% 0.72 Prolifer- ative 64 WNK449.37% 14.07% 0.71 65 PRPF4B 49.36% 12.18% 0.71 66 MAPKAPK2 49.30%12.83% 0.70 67 MEK WT 49.29% 14.33% 0.70 (−) Control 68 PRPS1 49.28%12.09% 0.70 69 BTK 49.22% 14.59% 0.69 70 MYO3B 49.19% 11.05% 0.69 71TESK1 49.18% 12.19% 0.68 72 PLXNA3 49.11% 9.04% 0.67 73 CLK2 49.09%8.01% 0.67 74 PFKFB1 49.02% 14.04% 0.66 75 DAK 49.00% 12.68% 0.66 76ITPKB 48.99% 16.62% 0.66 77 CHEK1 48.98% 14.41% 0.66 78 MERTK 48.94%12.88% 0.65 79 IKBKE 48.83% 12.46% 0.63 80 CARD11 48.83% 15.02% 0.63 81PANK3 48.78% 9.70% 0.63 82 TRIB2 48.76% 10.71% 0.63 83 CDC7 48.74% 9.83%0.62 84 PIK3C3 48.69% 12.05% 0.62 85 LRGUK 48.60% 11.29% 0.60 86 SCYL348.58% 10.86% 0.60 87 MAP3K14 48.57% 8.10% 0.60 88 LOC730000 48.57%13.36% 0.60 89 LOC442075 48.55% 10.75% 0.60 90 LOC54103 48.50% 10.77%0.59 91 NPR2 48.49% 13.29% 0.59 92 COL4A3BP 48.49% 11.10% 0.59 93 MYLK248.49% 11.83% 0.59 94 CSNK2B 48.48% 11.52% 0.59 95 CARKL 48.47% 9.19%0.58 96 TP53RK 48.44% 14.25% 0.58 Prolifer- ative 97 PRKCG 48.42% 8.31%0.58 98 ALK 48.39% 14.07% 0.57 99 ETNK1 48.37% 11.25% 0.57 100 DYRK248.36% 11.80% 0.57 101 DGKA 48.31% 10.71% 0.56 102 ADPGK 48.30% 13.36%0.56 103 PRKAB2 48.29% 10.75% 0.56 104 ULK3 48.28% 11.18% 0.56 105KIAA2002 48.28% 12.39% 0.56 106 IRAK3 48.26% 10.96% 0.56 107 TYK2 48.24%10.88% 0.55 108 MAP2K7 48.23% 12.06% 0.55 109 NRBP 48.18% 13.40% 0.54110 CDK2 48.14% 14.65% 0.54 111 MORN2 47.98% 11.36% 0.52 112 EPHB447.92% 12.52% 0.51 113 PRKCZ 47.88% 12.68% 0.50 114 RFK 47.85% 10.44%0.50 115 RAGE 47.83% 10.46% 0.50 116 LOC91807 47.80% 11.25% 0.49 117PIK3CG 47.76% 11.25% 0.49 118 PIK3C2G 47.74% 10.75% 0.48 119 ARSG 47.71%10.26% 0.48 120 CSNK1A1L 47.66% 10.26% 0.47 121 ALS2CR7 47.65% 11.56%0.47 122 FLJ40852 47.62% 12.34% 0.47 123 LOC390877 47.61% 11.43% 0.46124 TRIB1 47.59% 12.91% 0.46 125 TPR 47.58% 13.00% 0.46 126 RPS6KL147.57% 12.33% 0.46 127 CCRK 47.56% 10.28% 0.46 128 PDPK1 47.53% 10.42%0.45 129 FASTKD5 47.52% 10.70% 0.45 130 CDKL3 47.49% 13.14% 0.45 131DTYMK 47.49% 10.60% 0.45 132 MPP3 47.47% 10.65% 0.45 133 HSPB8 47.46%11.73% 0.44 134 NME6 47.44% 14.48% 0.44 135 NEK3 47.42% 15.46% 0.44 136PIK3R4 47.41% 11.14% 0.44 137 MGC16169 47.41% 13.90% 0.44 138 OXSR147.40% 11.68% 0.44 139 MASTL 47.35% 10.28% 0.43 140 PNCK 47.35% 10.15%0.43 141 ADCK2 47.32% 15.57% 0.43 142 SNX16 47.26% 12.76% 0.42 143 HCK47.24% 23.41% 0.41 144 CDKL2 47.21% 10.60% 0.41 145 NEK11 47.20% 14.68%0.41 146 BMP2K 47.15% 11.18% 0.40 147 BUB1 47.15% 11.76% 0.40 148 PINK147.13% 11.38% 0.40 149 RBKS 47.08% 8.73% 0.39 150 LOC732306 47.03% 8.82%0.39 151 PKLR 47.03% 12.10% 0.38 152 DYRK1A 47.01% 12.69% 0.38 153RPS6KA4 47.01% 11.70% 0.38 154 DGKG 46.94% 12.71% 0.37 155 CDK5R1 46.93%9.40% 0.37 156 FLJ25006 46.87% 11.64% 0.36 157 PBK 46.86% 14.05% 0.36158 ACVR1C 46.82% 9.98% 0.35 159 FLT1 46.78% 10.42% 0.35 160 PLXNA4B46.76% 9.39% 0.35 161 MVK 46.71% 9.26% 0.34 162 LIMK2 46.70% 11.29% 0.34163 DYRK1B 46.68% 20.75% 0.34 164 MARK3 46.67% 11.71% 0.34 Prolifer-ative 165 BMPR1B 46.66% 12.24% 0.33 166 NUP62 46.63% 9.92% 0.33 167 JAK246.62% 12.50% 0.33 168 MAPK12 46.55% 9.98% 0.32 169 DDR2 46.54% 7.99%0.32 170 MAK 46.52% 15.42% 0.31 171 GLYCTK 46.50% 13.19% 0.31 172 AK146.47% 9.68% 0.31 173 MAPK15 46.47% 11.14% 0.31 174 MAST1 46.45% 10.77%0.30 175 PAPSS2 46.39% 11.53% 0.30 176 CSF1R 46.34% 12.86% 0.29 177 TPK146.29% 9.52% 0.28 178 PAK4 46.24% 9.98% 0.28 179 NAGK 46.21% 12.34% 0.27180 CDK8 46.20% 7.68% 0.27 181 STK40 46.19% 12.92% 0.27 182 CIB1 46.10%10.92% 0.26 183 PLK1 46.08% 9.99% 0.25 184 FLJ23356 46.04% 10.45% 0.25185 LOC220686 46.04% 9.31% 0.25 186 PRKAB1 46.03% 12.00% 0.25 187 JAK346.02% 10.17% 0.24 188 NME1 46.02% 13.01% 0.24 189 NME5 46.00% 11.53%0.24 190 FER 45.97% 9.92% 0.24 191 AK3L1 45.97% 10.39% 0.24 192 PRKG245.90% 13.24% 0.23 193 RP6-213H19.1 45.88% 12.83% 0.22 194 AKT3 45.87%11.32% 0.22 195 PSKH1 45.86% 10.16% 0.22 196 PRKAR2B 45.84% 13.05% 0.22197 FUK 45.81% 10.94% 0.22 198 ADCK4 45.79% 11.25% 0.21 199 TEC 45.78%11.46% 0.21 200 PRKAG1 45.77% 11.08% 0.21 201 FXN 45.77% 12.03% 0.21 202AAK1 45.68% 11.49% 0.20 203 CAMK2B 45.67% 19.84% 0.20 204 COASY 45.64%11.07% 0.19 205 PRKAG3 45.63% 10.21% 0.19 206 NME7 45.58% 10.18% 0.18207 LMTK2 45.58% 12.15% 0.18 208 PANK2 45.57% 11.72% 0.18 209 PRKD345.47% 13.14% 0.17 210 PHKA2 45.47% 10.92% 0.17 211 SLAMF6 45.46% 9.67%0.17 212 SRPK1 45.46% 12.54% 0.17 213 HIPK1 45.42% 12.59% 0.16 214 EPHA145.39% 10.59% 0.16 215 WNK1 45.38% 13.79% 0.15 216 PDIK1L 45.33% 12.31%0.15 217 BMP2KL 45.33% 10.08% 0.15 218 MAP3K5 45.32% 14.13% 0.15 219EPHA2 45.30% 12.87% 0.14 220 CCL2 45.29% 9.71% 0.14 221 CDKL1 45.25%10.13% 0.14 222 NJMU-R1 45.23% 8.13% 0.13 223 LOC652799 45.18% 10.30%0.13 224 GUK1 45.15% 12.23% 0.12 225 NME4 45.12% 13.03% 0.12 226 YSK445.11% 13.47% 0.12 227 NEK2 45.11% 9.38% 0.12 228 C9orf95 45.10% 9.38%0.12 229 CDC2 45.05% 11.95% 0.11 230 FGFR2 45.01% 11.23% 0.10 231 IPMK44.98% 10.67% 0.10 232 STK32C 44.98% 12.10% 0.10 233 PIP5K2A 44.92%14.55% 0.09 234 PRKX 44.90% 10.44% 0.09 235 TRPM7 44.80% 10.36% 0.07 236FLJ10986 44.74% 10.95% 0.07 237 SNF1LK 44.74% 12.70% 0.07 238 MAP3K644.71% 9.75% 0.06 239 LOC653052 44.70% 14.56% 0.06 240 IRAK2 44.69%11.61% 0.06 241 XYLB 44.68% 11.47% 0.06 242 PTK7 44.67% 14.55% 0.06 243PKMYT1 44.66% 13.74% 0.05 244 SPHK2 44.63% 10.35% 0.05 245 NME2 44.63%11.57% 0.05 246 PRKAA1 44.62% 15.03% 0.05 247 MAPK14 44.60% 10.77% 0.05248 NTRK3 44.58% 13.43% 0.04 249 PRKACB 44.58% 9.30% 0.04 250 LOC65012244.56% 11.24% 0.04 251 CDK6 44.51% 10.74% 0.03 252 AMHR2 44.48% 11.11%0.03 253 IPPK 44.42% 10.63% 0.02 254 AK7 44.41% 10.36% 0.02 255 PIP5KL144.36% 11.74% 0.01 256 LOC340371 44.30% 16.04% 0.01 257 DKFZp434B123144.29% 12.21% 0.00 258 PRKAA2 44.28% 11.42% 0.00 259 FASTKD1 44.28%10.73% 0.00 260 ARAF 44.27% 14.40% 0.00 RAF Family 261 HK3 44.26% 14.83%0.00 262 KSR2 44.25% 10.28% 0.00 263 PAK7 44.24% 18.45% 0.00 264 GTF2H144.23% 10.90% 0.00 265 RPS6KB2 44.23% 9.32% −0.01 266 PAK6 44.21% 13.83%−0.01 267 IRAK4 44.18% 10.27% −0.01 268 NLK 44.18% 12.92% −0.01 269 FYN44.14% 12.43% −0.02 270 BMPR2 44.14% 12.20% −0.02 271 CDK4 44.13% 10.47%−0.02 272 STK32A 44.07% 13.93% −0.03 273 TNK1 44.06% 10.80% −0.03 274STK24 43.99% 10.79% −0.04 275 CSNK2A1 43.99% 11.97% −0.04 276 AK3 43.98%11.16% −0.04 277 TTK 43.93% 10.47% −0.05 278 PGK2 43.91% 13.31% −0.05279 GALK1 43.89% 10.14% −0.05 280 DYRK3 43.89% 9.32% −0.05 281 EIF2AK443.83% 11.00% −0.06 282 PDK1 43.79% 13.31% −0.07 283 EIF2AK1 43.79%12.02% −0.07 284 PRKACG 43.79% 11.37% −0.07 285 BUB1B 43.74% 10.69%−0.07 286 PKM2 43.74% 14.46% −0.07 287 LRPPRC 43.64% 10.87% −0.09 288EPHA6 43.63% 12.60% −0.09 289 HK2 43.58% 12.55% −0.10 290 MET 43.55%10.99% −0.10 291 CKS2 43.53% 7.83% −0.10 292 LOC375133 43.53% 10.05%−0.10 293 LIMK1 43.52% 12.43% −0.10 294 TWF2 43.50% 11.71% −0.11 295PCTK2 43.47% 12.02% −0.11 296 DCAKD 43.43% 11.61% −0.12 297 PLXNB243.41% 11.85% −0.12 298 MST1R 43.35% 11.99% −0.13 299 PRKAR1A 43.35%9.03% −0.13 300 SEPHS2 43.32% 9.68% −0.13 301 SPHK1 43.31% 9.87% −0.13302 CCL4 43.30% 11.83% −0.13 303 KIAA0999 43.28% 9.68% −0.14 304 PDGFRL43.23% 11.93% −0.14 305 BRSK1 43.21% 14.17% −0.15 306 BRAF 43.21% 12.56%−0.15 RAF Family 307 MUSK 43.20% 11.03% −0.15 308 TNNI3K 43.20% 10.46%−0.15 309 GK2 43.18% 15.15% −0.15 310 CKB 43.14% 9.45% −0.16 311 DGKB43.12% 11.62% −0.16 312 LOC648152 42.99% 11.15% −0.18 313 RPS6KA5 42.87%11.23% −0.19 314 CASK 42.87% 12.05% −0.19 315 PHKA1 42.86% 10.10% −0.20316 AK2 42.84% 9.83% −0.20 317 PIM1 42.81% 14.09% −0.20 318 ZAP70 42.81%12.10% −0.20 319 PNKP 42.79% 12.95% −0.20 320 CDK10 42.78% 9.77% −0.21321 CHEK2 42.77% 12.86% −0.21 322 CAMK2A 42.72% 11.68% −0.21 323 CAMK2G42.70% 9.66% −0.22 324 ADRBK2 42.70% 12.56% −0.22 325 NEK8 42.69% 9.88%−0.22 326 PRKR 42.69% 10.65% −0.22 327 CHKA 42.66% 12.84% −0.22 328ACVRL1 42.65% 10.85% −0.22 329 PRKY 42.63% 11.80% −0.23 330 TRIB3 42.62%11.45% −0.23 331 PRKD2 42.59% 10.48% −0.23 332 PIP5K3 42.57% 9.78% −0.24333 LOC727761 42.56% 14.17% −0.24 334 PTK2B 42.50% 15.72% −0.25 335 MPP242.48% 10.73% −0.25 336 CSNK1A1 42.47% 13.54% −0.25 337 PTK9 42.42%11.31% −0.26 338 STK25 42.41% 12.54% −0.26 339 PFKL 42.39% 12.01% −0.26340 STK19 42.35% 11.20% −0.27 341 PI4KII 42.30% 14.04% −0.27 342 HK142.26% 12.73% −0.28 343 AURKA 42.21% 14.80% −0.29 344 GK5 42.21% 10.98%−0.29 345 MAP3K7 42.20% 13.13% −0.29 346 PFTK1 42.18% 12.49% −0.29 347ERN1 42.17% 13.74% −0.29 348 STK33 42.17% 13.22% −0.29 349 SYK 42.13%12.42% −0.30 350 GALK2 42.13% 11.34% −0.30 351 TSSK1 42.12% 11.98% −0.30352 MAPK6 42.11% 10.58% −0.30 353 ASCIZ 42.07% 10.61% −0.30 354 PFKP42.07% 14.99% −0.31 355 PXK 42.05% 14.69% −0.31 356 PI4K2B 42.05% 11.84%−0.31 357 PIP5K2C 42.02% 9.50% −0.31 358 PFKFB3 42.00% 12.79% −0.31 359TSSK3 41.97% 14.82% −0.32 360 MPP6 41.97% 12.68% −0.32 361 MPP4 41.87%12.27% −0.33 362 LOC653155 41.85% 11.50% −0.34 363 ALPK1 41.82% 12.37%−0.34 364 CDK9 41.76% 10.32% −0.35 365 PDK3 41.66% 10.87% −0.36 366CKMT2 41.66% 12.57% −0.36 367 CAMK1G 41.63% 12.06% −0.37 368 MAPKAPK341.61% 13.19% −0.37 369 PIK4CB 41.55% 14.29% −0.38 370 PRPS1L1 41.54%11.08% −0.38 371 FASTK 41.49% 12.56% −0.39 372 CAMK1D 41.49% 11.54%−0.39 373 MARK2 41.48% 14.28% −0.39 374 PDK4 41.43% 13.43% −0.39 375NEK7 41.36% 9.57% −0.40 376 MAPK4 41.34% 10.83% −0.41 377 PIK4CA 41.32%11.99% −0.41 378 JAK1 41.31% 11.56% −0.41 379 PDXK 41.30% 11.93% −0.41380 TGFBR2 41.25% 15.85% −0.42 381 PHKB 41.25% 10.81% −0.42 382 ULK241.24% 15.35% −0.42 383 MKNK1 41.21% 13.08% −0.42 384 CDC2L6 41.17%10.91% −0.43 385 CSNK1G3 41.13% 10.53% −0.44 386 CAMK1 41.08% 11.68%−0.44 387 DGKZ 41.07% 12.69% −0.44 388 SGK 40.91% 11.92% −0.47 389 TBK140.83% 14.81% −0.48 390 ILK 40.81% 12.62% −0.48 391 STK32B 40.80% 11.79%−0.48 392 TXNDC3 40.78% 12.24% −0.48 393 RPS6KB1 40.72% 10.13% −0.49 394ZAK 40.71% 10.32% −0.49 395 DYRK4 40.66% 12.59% −0.50 396 ITGB1BP340.57% 11.99% −0.51 397 MPP1 40.53% 10.94% −0.52 398 HIPK2 40.49% 11.06%−0.52 399 MAPK13 40.46% 12.06% −0.53 400 TK1 40.39% 11.04% −0.54 401SH3BP4 40.36% 12.11% −0.54 402 CKM 40.29% 12.70% −0.55 403 FGFRL1 40.23%16.94% −0.56 404 MAPK10 40.23% 12.29% −0.56 405 CALM2 40.16% 8.64% −0.57406 CALM3 40.12% 11.85% −0.58 407 STYK1 40.09% 11.47% −0.58 408 CDKL440.08% 11.01% −0.58 409 NADK 40.08% 11.89% −0.58 410 MPP7 40.06% 11.72%−0.59 411 CAMKV 40.03% 12.16% −0.59 412 EXOSC10 40.02% 12.78% −0.59 413CDKL5 39.98% 10.10% −0.60 414 STK38 39.90% 9.61% −0.61 415 INSRR 39.90%15.08% −0.61 416 DCK 39.86% 10.23% −0.61 417 TLK2 39.80% 14.66% −0.62418 PCTK3 39.66% 10.48% −0.64 419 LOC388957 39.65% 10.97% −0.64 420PAPSS1 39.59% 13.48% −0.65 421 ACVR2B 39.56% 14.70% −0.65 422 NME1-NME239.53% 12.01% −0.66 423 NEK10 39.50% 13.78% −0.66 424 MAP3K11 39.47%9.33% −0.67 425 PMVK 39.46% 10.19% −0.67 426 MAPK9 39.45% 12.74% −0.67427 MKNK2 39.39% 13.54% −0.68 428 GRK7 39.34% 14.98% −0.68 429 RIOK239.27% 11.79% −0.69 430 DGKK 39.27% 9.02% −0.69 431 ACVR1 39.15% 13.96%−0.71 432 TLK1 39.11% 15.46% −0.72 433 LATS1 39.05% 10.46% −0.73 434SCYL1 39.01% 11.41% −0.73 435 TESK2 38.93% 13.30% −0.74 436 DGUOK 38.90%14.54% −0.75 437 PGK1 38.87% 12.26% −0.75 438 MAGI1 38.87% 12.66% −0.75439 SNRK 38.79% 11.61% −0.76 440 CALM1 38.65% 10.26% −0.78 441 RIOK138.58% 12.39% −0.79 442 EEF2K 38.56% 11.96% −0.79 443 MAPK8 38.45%10.51% −0.81 444 CSNK1D 38.44% 15.60% −0.81 445 ULK4 38.42% 12.02% −0.81446 STK38L 38.33% 12.63% −0.83 447 RIOK3 38.21% 12.80% −0.84 448 MINK138.00% 14.94% −0.87 449 ROR2 37.95% 15.32% −0.88 450 PTK2 37.93% 10.59%−0.88 451 PIK3R5 37.84% 13.17% −0.89 452 ALDH18A1 37.81% 15.66% −0.90453 NYD-SP25 37.79% 13.52% −0.90 454 MAP4K3 37.76% 9.58% −0.90 455 AGK37.61% 13.89% −0.92 456 GSK3A 37.56% 14.44% −0.93 457 BMPR1A 37.56%16.10% −0.93 458 STK16 37.53% 11.09% −0.94 459 FASTKD2 37.50% 8.53%−0.94 460 MAP4K4 37.39% 15.68% −0.96 461 LRRK2 37.38% 10.11% −0.96 462TGFBR3 37.33% 11.04% −0.96 463 PDGFRB 37.29% 16.90% −0.97 464 DLG337.09% 12.08% −1.00 465 PFKFB2 36.99% 10.37% −1.01 466 AKT1 36.66%13.43% −1.06 467 PRKCI 36.54% 11.44% −1.07 468 NEK4 36.50% 13.19% −1.08469 PRKD1 36.45% 14.57% −1.09 470 SRPK2 36.37% 12.38% −1.10 471 SH3BP536.37% 17.17% −1.10 472 CLK1 36.06% 11.13% −1.14 473 GK 35.86% 17.33%−1.17 474 IHPK1 35.53% 12.15% −1.21 475 IHPK3 35.48% 9.30% −1.22 476PLAU 35.36% 13.10% −1.24 477 TAOK3 35.29% 14.78% −1.25 478 PAK2 35.20%12.51% −1.26 479 BMX 35.12% 12.94% −1.27 480 DAPK2 35.01% 13.92% −1.29481 CSNK1E 34.83% 15.21% −1.31 482 MAPK3 34.75% 19.93% −1.32 Lethal 483MAP3K15 34.40% 16.76% −1.37 Lethal 484 MPP5 34.35% 11.80% −1.38 485MAST2 34.08% 16.10% −1.42 486 GRK6 34.04% 15.82% −1.42 487 DKFZp761P042333.93% 13.58% −1.44 488 VRK1 33.87% 9.28% −1.45 489 DCAMKL2 33.77%11.96% −1.46 490 IHPK2 33.75% 9.56% −1.46 491 MATK 33.44% 10.25% −1.51492 RP2 33.20% 13.41% −1.54 493 MAP2K2 32.98% 15.34% −1.57 494 UCK232.82% 13.75% −1.59 495 NEK6 32.74% 15.85% −1.60 496 PRKG1 32.52% 14.60%−1.63 497 MAP3K12 32.49% 15.33% −1.64 498 PCTK1 32.48% 13.21% −1.64 499MGC42105 32.36% 13.85% −1.66 500 MAP2K6 32.32% 11.92% −1.66 501 SH3BP5L32.11% 15.55% −1.69 502 FES 32.07% 11.04% −1.70 503 RKHD3 31.89% 12.45%−1.72 504 PRKACA 31.76% 18.45% −1.74 Lethal 505 MAP4K1 31.51% 13.69%−1.77 506 CSNK1G1 30.32% 14.90% −1.94 507 MAPKAPK5 30.15% 13.47% −1.96508 CSK 29.83% 14.11% −2.01 Lethal 509 BRD3 29.16% 8.83% −2.10 510ADRBK1 29.00% 16.93% −2.12 511 BRSK2 28.64% 13.09% −2.17 512 ADCK128.30% 13.52% −2.22 513 CSNK1G2 26.81% 14.42% −2.43 514 CKMT1A 26.80%12.42% −2.43 515 TSSK2 25.66% 13.30% −2.59 Lethal 516 CD2 25.01% 14.99%−2.68 517 PIP5K1A 24.76% 15.49% −2.71 518 PHKG1 23.31% 14.24% −2.91 519ABL2 Low pLX DNA yield 520 ACVR2A Low pLX DNA yield 521 BLK Low pLX DNAyield 522 CDC2L1 Low pLX DNA yield 523 EGFR Low pLX DNA yield 524 EPHA3Low pLX DNA yield 525 EPHB1 Low pLX DNA yield 526 ERBB4 Low pLX DNAyield 527 FASTKD3 Low pLX DNA yield 528 FGFR1 Low pLX DNA yield 529 FLT3Low pLX DNA yield 530 FLT4 Low pLX DNA yield 531 HIPK3 Low pLX DNA yield532 KSR Low pLX DNA yield 533 LYN Low pLX DNA yield 534 PANK4 Low pLXDNA yield 535 PDGFRA Low pLX DNA yield 536 PLK4 Low pLX DNA yield 537RET Low pLX DNA yield 538 SGK3 Low pLX DNA yield 539 SRC Low pLX DNAyield 540 TXK Low pLX DNA yield 541 BRD4 Inf. Effic. <70% 542 CAMKK2Inf. Effic. <70% 543 CKS1B Inf. Effic. <70% 544 CLK3 Inf. Effic. <70%545 DAPK3 Inf. Effic. <70% 546 EPHA4 Inf. Effic. <70% 547 EPHB6 Inf.Effic. <70% 548 FGFR3 Inf. Effic. <70% 549 FRK Inf. Effic. <70% 550 GCKInf. Effic. <70% 551 GNE Inf. Effic. <70% 552 HIPK4 Inf. Effic. <70% 553ITK Inf. Effic. <70% 554 KDR Inf. Effic. <70% 555 LOC389599 Inf. Effic.<70% 556 LOC649288 Inf. Effic. <70% 557 MAP3K2 Inf. Effic. <70% 558 NEK5Inf. Effic. <70% 559 NTRK1 Inf. Effic. <70% 560 PAK1 Inf. Effic. <70%561 PHKG2 Inf. Effic. <70% 562 PIK3CA Inf. Effic. <70% 563 PIK3R3 Inf.Effic. <70% 564 PIP5K1B Inf. Effic. <70% 565 RIPK1 Inf. Effic. <70% 566RIPK2 Inf. Effic. <70% 567 RPS6KA2 Inf. Effic. <70% 568 RPS6KC1 Inf.Effic. <70% 569 STK17B Inf. Effic. <70% 570 TGFBR1 Inf. Effic. <70% 571UHMK1 Inf. Effic. <70% 572 XRCC6BP1 Inf. Effic. <70% 573 BCKDK ReplicateSTDEV 574 C1orf57 Replicate STDEV 575 CAMKK1 Replicate STDEV 576 CDC2L2Replicate STDEV 577 CDK5 Replicate STDEV 578 ERBB3 Replicate STDEV 579GSG2 Replicate STDEV 580 LOC647279 Replicate STDEV 581 LYK5 ReplicateSTDEV 582 MAPK1 Replicate STDEV Lethal 583 MAP2K5 Replicate STDEV 584MAP4K2 Replicate STDEV 585 NUAK2 Replicate STDEV 586 PDK2 ReplicateSTDEV Lethal 587 PFKM Replicate STDEV 588 PIK3R1 Replicate STDEV 589PLK2 Replicate STDEV 590 PRKAR2A Replicate STDEV 591 RPS6KA3 ReplicateSTDEV 592 RPS6KA6 Replicate STDEV 593 SGK2 Replicate STDEV 594 SRPK3Replicate STDEV 595 VRK1 Replicate STDEV 596 VRK2 Replicate STDEV 597VRK3 Replicate STDEV A375 SKMEL28

TABLE 5 Results of a secondary screen quantifying the change in PLX4720GI₅₀ induced by the top 9 candidate resistance ORFs Secondary ScreenA375 SKMEL28 GI₅₀ Fold Change GI50 Fold Change Gene (μM) GI₅₀ Rank Gene(μM) GI₅₀ Rank MAP3K8 >100.0 598 1 MAP3K8 >100.0 ~100 1 RAF1 ≧100.0 5982 RAF1 ≧10.0 ≧10 2 CRKL >10.0 59.8 3 CRKL 9.7 9.7 3 FGR >10.0 59.8 4 FGR5 5 4 PRKCE 4.41 26.4 5 PRKCH 2.26 2.26 5 PRKCH 4.14 24.7 6 PRKCE 1.911.91 6 ERBB2 1.33 7.95 7 AXL 1.18 1.18 7 AXL 1 5.98 8 ERBB2 1 1 8 PAK30.4934 2.95 9 PAK3 0.9041 0.9041 9 Controls Controls Mock 0.1528 0.91Mock 0.5287 1.89 MEK1 0.1671 1 (−) MEK1 1 1 (−) MEK DD 4.8 28 (+) MEK DD8.29 8.29 (+)

TABLE 6 Patient characteristics BRAF Time Time from status frommetastatic BRAF (on- primary disease status treatment/ Age Biopsydiagnosis diagnosis (pre- post- NRAS KRAS pMEK pERK Patient Tissue(years) Gender Location (years) (years) Response treatment) relapse)status status status status Pt 1 Tissue 49 F Back 5 3 Partial V600E N/AN/A N/A N/A N/A Biopsy Response Pt 2 Tissue 67 M Back 2 0 Partial V600EN/A N/A N/A N/A N/A Biopsy Response Pt 3 Tissue 68 M Leg 11 0 PartialV600E N/A N/A N/A N/A N/A Biopsy Response MM-R Tissue 38 M Axilla 0 0Partial V600E V600E WT WT * * Biopsy Response M307 Short- 59 M Axillary4 4 Stable V600E V600E N/A N/A Ref. ** term Lymph Disease (14) Culturenode * See FIG. 20 ** See FIG. 3f

1. A method of identifying a subject having cancer who is likely tobenefit from treatment with a combination therapy with a RAF inhibitorand a second inhibitor, comprising: (a) assaying a gene copy number, amRNA or a protein level or phosphorylation of one or more kinase targetsselected from the group consisting of MAP3K8 (TPL2/COT), RAF1 (CRAF),CRKL (CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL(Axl), or PAK3 (Pak3) in cancer cells obtained from the subject andcomparing the gene copy number, the mRNA or the protein level or thephoshorylation with a gene copy number, a mRNA or a protein level orphosphorylation of the target kinase in cells obtained from a subjectwithout the cancer; (b) correlating increased gene copy number or analteration in mRNA expression or protein overexpression orphosphorylation of the target kinase in the cancer cells relative to thecells from the subject without the cancer with the subject having thecancer who is likely to benefit from treatment with the combinationtherapy; and (c) selecting the subject having increased gene copy numberor an alteration in mRNA expression or protein overexpression orphosphorylation of the target kinase in the cancer cells for treatmentwith the combination therapy.
 2. The method of claim 1, furthercomprising (c) assaying a nucleic acid sample obtained from the cancercells for the presence of a mutation in a nucleic acid molecule encodinga B-RAF polypeptide with a mutation at about amino acid position 600 and(d) correlating the presence of the mutation in the nucleic acidmolecule encoding the B-RAF polypeptide with a subject who is likely tobenefit from treatment with the combination therapy.
 3. The method ofclaim 1, comprising correlating the presence of a Valine to GlutamicAcid mutation at about amino acid position 600 (V600E) in a nucleic acidmolecule encoding B-RAF with a subject who is likely to benefit fromtreatment with the combination therapy.
 4. The method of claim 1,comprising assaying the gene copy number, the mRNA or the protein levelof MAP3K8 (TPL/COT).
 5. The method of claim 1, comprising assayingphosphorylation of C-RAF at amino acid S338.
 6. The method of claim 1,wherein the second inhibitor is a MEK inhibitor, a CRAF inhibitor, aCrkL inhibitor or a TPL2/COT inhibitor.
 7. The method of claim 1,wherein the RAF inhibitor is a B-RAF inhibitor or a pan-RAF inhibitor.8. The method of claim 1, wherein the RAF inhibitor is selected from thegroup consisting of RAF265, sorafenib, SB590885, PLX 4720, PLX4032,GDC-0879 and ZM
 336372. 9. The method of claim 1, wherein the subjecthas innate resistance to the RAF inhibitor or is likely to developresistance to the RAF inhibitor.
 10. The method of claim 1, wherein thecancer is selected from the group consisting of melanoma, breast cancer,colorectal cancers, glioma, lung cancer, ovarian cancer, sarcoma andthyroid cancer.
 11. The method of claim 10, wherein the cancer ismelanoma.
 12. A method of treating cancer in a subject in need thereof,comprising administering to the subject an effective amount of a RAFinhibitor and an effective amount of a second inhibitor, wherein thesecond inhibitor is a MEK inhibitor, or a MAP3K8 (TPL2/COT) inhibitor.13. The method of claim 12, wherein the subject has cancer cellscomprising a BRAF mutation or a B-RAF^(V600E) mutation.
 14. (canceled)15. The method of claim 12, further comprising assaying a gene copynumber, a mRNA or a protein level or phosphorylation of one or morekinase targets selected from the group consisting of MAP3K8 (TPL2/COT),RAF1 (CRAF), CRKL (CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2(ErbB2), AXL (Axl), or PAK3 (Pak3) in cancer cells obtained from thesubject and comparing the gene copy number, the mRNA or the proteinlevel or the phoshorylation with a gene copy number, a mRNA or a proteinlevel or phosphorylation of the target kinase in cells obtained from asubject without the cancer; and administering to the subject theeffective amount of a RAF inhibitor and the effective amount of a secondinhibitor in subjects having an increased gene copy number or analteration in mRNA expression or protein overexpression orphosphorylation of the target kinase in the cancer cells.
 16. The methodof claim 12, wherein the RAF inhibitor is selected from the groupconsisting of RAF265, sorafenib, SB590885, PLX 4720, PLX4032, GDC-0879and ZM
 336372. 17. The method of claim 12, wherein the MEK inhibitor isselected from the group consisting of CI-1040/PD184352, AZD6244,PD318088, PD98059, PD334581, RDEA119,6-Methoxy-7-(3-morpholin-4-yl-propoxy)-4-(4-phenoxy-phenylamino)-quinoline-3-carbonitrileand4-[3-Chloro-4-(1-methyl-1H-imidazol-2-ylsulfanyl)-phenylamino]-6-methoxy-7-(3-morpholin-4-yl-propoxy)-quinoline-3-carbonitrile.18. The method of claim 12, wherein the subject has innate resistance tothe RAF inhibitor or the MEK inhibitor or is likely to developresistance to the RAF inhibitor or the MEK inhibitor.
 19. The method ofclaim 12, wherein the cancer is selected from the group consisting ofmelanoma, breast cancer, colorectal cancers, glioma, lung cancer,ovarian cancer, sarcoma and thyroid cancer.
 20. The method of claim 12,wherein the cancer is melanoma.
 21. (canceled)
 22. The method of claim12, wherein the second inhibitor is a MAP3K8 (TPL2/COT) inhibitor.
 23. Amethod of identifying a kinase target that confers resistance to a firstinhibitor, the method comprising: culturing cells having sensitivity tothe first inhibitor; expressing a plurality of kinase ORF clones in thecell cultures, each cell culture expressing a different kinase ORFclone; exposing each cell culture to the inhibitor; identifying cellcultures having greater viability than a control cell culture afterexposure to the inhibitor to identify one or more kinase ORF clones thatconfers resistance to the first inhibitor.
 24. The method of claim 23,wherein the cultured cells have sensitivity to a RAF inhibitor or a MEKinhibitor.
 25. (canceled)
 26. The method of claim 23, wherein thecultured cells comprise a B-RAF mutation or a B-RAF^(V600E) mutation.27. (canceled)
 28. The method of claim 23, wherein the cultured cellscomprise a melanoma cell line.
 29. The method of claim 23, comprisingproviding the plurality of kinase ORF clones in a lentiviral vector or aretroviral vector.